Circadian rhythm of dihydrouracil/uracil ratios in biological fluids: a potential biomarker for dihydropyrimidine dehydrogenase levels

Abstract

1. In many cancer patients, 5-fluorouracil (5-FUra) treatment is toxic and even causes death. Nevertheless, all patients are subjected to a standard therapy regimen because there is no reliable way to identify beforehand those patients who are predisposed to 5-FUra-induced toxicity. In this study, we identified the dihydrouracil/uracil (UH2/Ura) ratio in plasma or urine as a potential biomarker reflecting the activity of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in 5-FUra metabolism. 2. UH2/Ura ratios were measured by high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-MS/MS) in both healthy subjects (n=55) and in patients (n=20) diagnosed with grade I/II gestational trophoblastic tumours. In addition, rats (n=18) were used as an animal model to verify a correlation between UH2/Ura ratios and DPD levels in the liver. 3. A significant circadian rhythm was observed in UH2/Ura ratios in healthy subjects, whereas a disrupted rhythm occurred in cancer patients who were continuously infused with a high dose of 5-FUra. In rats, UH2/Ura ratios, liver DPD levels and PBMC DPD levels showed a definite circadian rhythm. Significant linear correlations with liver DPD levels were demonstrated for plasma UH2/Ura ratios (r=0.883, P<0.01), urine UH2/Ura ratios (r=0.832, P<0.01) and PBMC DPD levels (r=0.859, P<0.01). 4. The UH2/Ura ratio in biological fluid was significantly correlated with liver DPD levels; hence, this ratio could be a potential biomarker to identify patients with a deficiency in DPD.

Figure 1

Circadian rhythms of UH2/Ura ratios in human plasma (a, n=12), human urine (b, n=55) and DPD levels in human PBMCs (c, n=12). Time of awakening was between 06:30 and 07:00 h and that of retiring was between 21:30 and 22:00 h. The curves were computer-generated and analysed by ‘Cosinor' analysis, x±s.d.

Figure 2

Linear correlations between human PBMC DPD levels and UH2/Ura ratios in plasma (a) and urine (b) at eight different time points during the day. The mean value at each time point was obtained from 12 randomly selected healthy subjects.

Figure 3

Comparison of the variance between 5-FUra concentrations and UH2/Ura ratios in patient plasma. Patients were continuously infused 5-FUra (30 mg kg⁻¹, n=20, x±s.d.) for 8 h starting from 09:00 h, and blood samples were collected at different time points, 09:30, 12:00, 15:00, 17:00, 17:10, 17:20 and 18:20 h.

Figure 4

Relationship between 5-FUra concentrations and plasma UH2/Ura ratios in the patients administered 5-FUra.

Figure 5

Circadian rhythms of UH2/Ura ratios in rat plasma (a), UH2/Ura ratios in rat urine (b) and DPD levels in rat liver(c) and PBMCs (d). Data were obtained from the study in rats under the following conditions: lights on from 06:00 to 18:00 h, and lights off from 18:00 to 06:00 h. Three animals were used for each time point. The curves were computer-generated and analysed by ‘Cosinor' analysis. In order to emphasize the relationship between circadian pattern and light–dark cycle, the data were plotted by setting the abscissa from 0–24 HALO. 0–12 HALO, the period when light was on; 12–24 HALO, the period when lights were off.

Figure 6

Linear correlations with rat liver DPD levels for plasma UH2/Ura ratios (a, r=0.883, P<0.01), urine UH2/Ura ratios (b, r=0.832, P<0.05) and PBMC DPD levels (c, r=0.859, P<0.01), n=18.