Specialized characterization using different cfDNA analyses of blood samples from an exercise intervention study with liver patients

Chronic liver diseases have been reported to go along with increased concentrations of circulating cell free DNA (cfDNA). Furthermore, acute and chronic exercise increases cfDNA levels in healthy subjects.

We took venous as well as capillary blood samples from 40 patients with inflammatory (NASH) as well as non-inflammatory (NAFLD) liver diseases before and directly after exhausting cycling tests and after 90 min of recovery, in the beginning and at the end of an 8 week exercise intervention study. To test for influences of sample processing, we centrifuged the venous blood samples either at 16000 xg or at 1600 xg. Plasma cfDNA was quantified directly from blood plasma for all samples by two qPCRs targeting either a short (90 bp) or a longer (222 bp) fragment of the L1PA2 locus.

No differences were observed between the different blood sample types. Furthermore, the concentrations of the longer fragments correlated well with the shorter fragments. In general, the patients with an inflammatory liver disease had significantly higher cfDNA levels compared to NAFLD-patients at rest and after recovery, but not directly after exercise, when the two groups did not differ. Mean resting but not recovery cfDNA concentrations in both groups increased over the intervention period, despite significant improvements of the disease symptoms and in physical fitness. Interestingly, resting cfDNA concentrations of the short fragments correlated with the disease markers before but not after the intervention. However, after the exercise test and the following recovery period, associations between cfDNA and the disease markers were restored. These associations were not detectable for the longer fragments.

Our results emphasize the importance of the timing and methodology of cfDNA analysis. A bias of resting concentrations induced by external influences such as chronic exercise can be reduced by transient increases and a following recovery period.