Changes in CoQ10/Lipids Ratio, Oxidative Stress, and Coenzyme Q10 during First-Line Cisplatin-Based Chemotherapy in Patients with Metastatic Urothelial Carcinoma (mUC)

Oxidative stress plays an important role in cancer pathogenesis, and thiobarbituric acid-reactive substance level (TBARS)—a parameter of lipid peroxidation—has prognostic significance in chemotherapy-naive patients with metastatic urothelial carcinoma (mUC). However, the effect of cisplatin (CDDP)-based chemotherapy on oxidative stress, coenzyme Q10, and antioxidants remains unknown. The objective of this prospective study was to determine possible changes in the CoQ10 (coenzyme Q10)/lipids ratio, antioxidants (α-tocopherol, γ-tocopherol, β-carotene, CoQ10), total antioxidant status (TAS), and TBARS in plasma at baseline and during first-line chemotherapy based on CDDP in mUC subjects. In this prospective study, 63 consecutive patients were enrolled. The median age was 66 years (range 39–84), performance status according to the Eastern Cooperative Oncology Group (ECOG) was 2 in 7 subjects (11.1%), and visceral metastases were present in 31 (49.2%) patients. Plasma antioxidants were determined by HPLC and TAS and TBARS spectrophotometrically. After two courses of chemotherapy, we recorded significant enhancements compared to baseline for total cholesterol (p < 0.0216), very low-density lipoprotein (VLDL) cholesterol (p < 0.002), triacylglycerols (p < 0.0083), α-tocopherol (p < 0.0044), and coenzyme Q10-TOTAL (p < 0.0001). Ratios of CoQ10/total cholesterol, CoQ10/HDL-cholesterol, and CoQ10/LDL-cholesterol increased during chemotherapy vs. baseline (p < 0.0048, p < 0.0101, p < 0.0032, respectively), while plasma TBARS declined (p < 0.0004). The stimulation of antioxidants could be part of the defense mechanism during CDDP treatment. The increased index of CoQ10-TOTAL/lipids could reflect the effect of CDDP protecting lipoproteins from peroxidation.


Introduction
Bladder cancer (BC) is the tenth most common cancer in the world, and its incidence is steadily rising worldwide [1]. Almost 90% of all BCs are urothelial carcinomas [2]. Patients with muscle-infiltrating BC (MIBC) account for about 30% of all cases. Despite considerable advances in systemic treatment of MIBC during recent years, cisplatin (CDDP) still remains a key agent [3], while up to 50% of patients fail to respond to CDDP-based chemotherapy [4]. Lymph nodes, lungs, liver, bones, and the peritoneum belong among the most common sites of distant metastases in subjects with metastatic urothelial carcinoma (mUC) [3]. The independent factors for predicting survival in mUC patients, as well as performance status assessment using the Eastern Cooperative Oncology Group (ECOG) and visceral metastasis, were established in the 1990s [5].

Plasma Lipids, Antioxidants, and Lipid Peroxidation Baseline and during Chemotherapy
When values after two courses of chemotherapy were compared to baseline, the changes were determined. There were significant increases in total cholesterol, VLDL-cholesterol, TGs, α-tocopherol, CoQ 10-TOTAL , CoQ 10-TOTAL /total cholesterol, CoQ 10-TOTAL /HDL-cholesterol, and CoQ 10-TOTAL /LDL-cholesterol, while TBARS levels declined. Other parameters did not change significantly (Table 1). Table 1. Plasma lipids, antioxidants, index CoQ 10-TOTAL /lipids, and oxidative stress during chemotherapy in metastatic urothelial carcinoma patients (mUC). N: number of patients; SD: standard deviation; SEM: standard error mean; HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low-density lipoprotein; TGs: triacylglycerols; CoQ 10 : coenzyme Q 10 ; TBARS: thiobarbituric acid-reactive substances; * significant. To identify whether changes in individual parameters are affected by performance status, we divided the population of mUC patients into ECOG 0-1 and ECOG 2 subgroups, and evaluated the differences using repeated-measure analysis of variance.
Except α-tocopherol, no significant changes were determined (see Table 2). In the next step, the study population was split into subgroups with visceral metastasis absent or present, and compared as described above. There was no significant difference in any parameter regarding visceral metastasis status (Table 3).

Plasma Lipids, Antioxidants, and Oxidative Stress Baseline and during Chemotherapy-A Subgroup Analysis by Objective Response
To perform this subgroup analysis, the study population was divided by objective (complete and partial) response (OR) to subgroups with absent and present OR. The changes in any of the explored parameters were not affected by OR in mUC patients treated with first-line CDDP-based combined chemotherapy.
Detailed data are shown in Table 4.  Finally, the study population was split into subgroups in which serious AEs were present or absent. As Table 5 shows, the dynamics of any parameter did not depend on serious AEs.

Discussion
In this prospective study, we revealed a significant increase in total cholesterol, VLDLcholesterol, and TGs after two courses of CDDP-based chemotherapy compared to baseline in patients with mUC (Table 1). In general, lipids were enhanced regardless of ECOG performance status, the presence (or absence) of visceral metastases, objective response (present or absent), and serious adverse events (present or absent) ( Tables 2-5).
Determining the effect of CDDP-based chemotherapy on plasma lipids was the objective of studies with germ cell tumor patients in the early 1990s. A study by Boyer et al. [23] revealed a significant elevation in serum cholesterol in subjects with metastatic germ cell tumors treated with CDDP-containing chemotherapy when compared to a control population. At the time of lipid measurement, all their patients were in complete remission. Similarly, a study conducted by Raghavan et al. [24] reported that hypercholesterolemia is one of the potential effects of CDDP-based chemotherapy for testicular cancer. However, Ellis et al. did not demonstrate an elevation in total plasma cholesterol after CDDP chemotherapy in a similar population of patients [25]; however, they hypothesized that alterations in plasma lipids could be the result of an enhanced production of cytokines, including tumor necrosis factor (TNF), and varied according to the extent of the disease [23].
After entering the key words "effect", "cisplatin", "lipids", "serum", "bladder", and "cancer" into the Pubmed database, we did not find any paper addressing this issue. This could, therefore, be first study to show the effect of CDDP on plasma lipid levels in patients with mUC. Since no patient in this study took lipid-lowering drugs, or smoked during treatment, and no significant changes in BMI were noticed, we believe that the identified changes in plasma lipid levels resulted from systemic therapy. Moreover, they did not depend on its effectiveness, patient performance status, visceral metastasis, or the presence of serious adverse events.
Our study showed a significant rise in both CoQ 10-TOTAL and α-tocopherol after CDDPbased chemotherapy vs. baseline in patients with mUC (Table 1). These changes did not depend on performance status, visceral metastasis, objective response, or serious AEs (Tables 2-5), except for α-tocopherol plasma levels, when the study population was split by performance status. However, this subgroup analysis must be interpreted with caution, as there were only seven patients with ECOG 2 ( Table 2).
Because CoQ 10 is a component of lipoproteins mainly present in the plasma, where about 75% is associated with LDL and the remaining is localized in blood cells (platelets, erythrocytes, and leucocytes) [26], we also calculated ratios of CoQ 10-TOTAL and lipids. CoQ 10-TOTAL /total cholesterol, CoQ 10-TOTAL /HDL-cholesterol, and CoQ 10-TOTAL /LDLcholesterol indexes significantly increased during chemotherapy compared to baseline. On the other hand, plasma levels of TBARS, a parameter of a lipid peroxidation, significantly declined during chemotherapy when compared to the baseline value ( Table 1). None of these changes were influenced by the performance status of the patients, the presence of visceral metastases before chemotherapy initiation, objective response achieved by the systemic treatment, or serious AEs related to CDDP-based chemotherapy (Tables 2-5).
CoQ 10 plasma levels were an independent prognostic factor that could be used to estimate the risk for pancreatic carcinoma [27] and melanoma progression [28]. Low plasma CoQ 10 was significantly associated with an increased risk of lung cancer, particularly among current smokers, and may be related to disease progression [29]. Matrix metalloproteinases 2 (MMP-2) plays a key role in cellular invasion and metastasis. Exogenous CoQ 10 reduces MMP-2 activity, along with the pro-oxidant capacity of cancer cells in a dose-proportionate manner. Mitochondrial ROS is the mediator of MMP-2 activity [30].
A prospective study by Slopovsky et al. [31] showed that low levels of a marker of lipid peroxidation-TBARS-detected in the plasma of chemotherapy-naive patients with mUC correlated with better progression-free survival (PFS) and overall survival (OS). In our previous study [32], the changes in platelet mitochondrial bioenergetics that are key for cell reprogramming in patients with UC were identified. We hypothesized that increased oxidative stress, decreased oxidative phosphorylation (OXPHOS), and a reduced endogenous CoQ 10 in platelets could contribute to the reprogramming of mitochondrial OX-PHOS towards the activation of glycolysis, impaired mitochondrial function, and increased oxidative stress by initiating reverse electron transport from CoQ 10 to complex I.
Based on the current study's results, we assume that there is an interaction between CoQ 10 and CDDP, as it has the ability to bind many hydrogens [13]. CDDP could be a Q-CYCLE proton donor leading to the stimulation of CoQ 10 production. An increased concentration of CoQ 10 can stimulate the transport of electrons from complex I and complex II to complex III, and increase mitochondrial ATP production through OXPHOS. Ubiquinone is reduced to ubiquinol, which is a stronger antioxidant, and lipid peroxidation is reduced. Raised CoQ 10 concentrations can regenerate α-tocopherol and enhance its level.
To conclude, during first-line CDDP-based chemotherapy in patients with mUC, a significant stimulation of lipid fractions (total cholesterol, VLDL-cholesterol, and TGs) and the production of antioxidants (CoQ 10-TOTAL and α-tocopherol) along with lipid peroxidation suppression were evident. The enhancement of cholesterol and TGs is not favorable, but the stimulation of antioxidants could represent a host defense mechanism during CDDP treatment. The increased index of CoQ 10-TOTAL /lipids could reflect the beneficial effect of CDDP in protecting lipoproteins from peroxidation. These findings contribute new insights into the effects of CDDP in patients with mUC.

Inclusion/Exclusion Criteria and Study Design
All subjects enrolled into this study met the following inclusion criteria: age ≥ 18 years, a diagnosis of MIBC or muscle-infiltrating urothelial carcinoma of the upper tract (the renal pelvis or ureter) confirmed histologically or cytologically, measurable disease based on RECIST 1.1 criteria, at least one distant metastasis, no prior chemotherapy for inoperable locally advanced or mUC, an ECOG performance status of 0, 1, or 2, and adequate organ function.
Exclusion criteria included disease suitable for local therapy administered with curative intent, a previous malignancy, other than basal or squamous cell carcinomas of the skin, progressing or requiring active treatment within the past 5 years or undergoing potentially curative therapy, in situ cervical cancer, known psychiatric disorders or substance abuse that could have interfered with cooperation with the requirements of this study, known regular use of any illicit drug or a recent history (within the past year) of drug or alcohol abuse, known history of human immunodeficiency virus (HIV), or active hepatitis B or hepatitis C. Concomitant medication with lipid-lowering drugs or triacylglycerol-lowering agents were further exclusion criteria.
We conducted a prospective, non-randomized, single-center observational study to explore specified outcomes outlined in the Introduction section. This study was approved by the Ethical Committee at the National Cancer Institute, Bratislava, Slovakia (protocol code: UC-SK001). All data were entered by investigators into electronic data files and their accuracy was validated for each patient by an independent investigator.

Characteristics of Patients
A total of 63 consecutive patients who met the eligibility requirements were enrolled into this prospective study conducted at the National Cancer Institute in Bratislava (Slovakia). Median age was 66 years (range 39-84 years), and the majority of subjects were male (N = 50, 79.4%). The primary tumor site was the bladder in 82.5% of cases and the upper urinary tract (renal pelvis or ureter) in 17.5%. All patients had pure urothelial carcinoma, and 90.5% of subjects had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1, and 9.5% scored 2. At least one visceral metastasis was present in 49.2% of all cases. Baseline median body mass index (BMI) was 29.6 kg/m 2 (range 20.4-34.5 kg/m 2 ) without a significant change at week 6.
All subjects were treated with cisplatin 70 mg/m 2 intravenously on day 1 with gemcitabine 1000 mg/m 2 intravenously on days 1 and 8. A new treatment cycle started on day 22. The total number of chemotherapy courses was 6. The effect of therapy was evaluated with RECIST 1.1 criteria. Complete response (CR) was recorded in 19.1% and partial response (PR) in 38.1%. The remaining subjects did not respond to systemic therapy.
Progression-free survival (PFS) was calculated from day 1 of the first course of chemotherapy until disease progression, last follow-up, or death from any cause. Overall survival (OS) was calculated from day 1 of the first course of chemotherapy until last follow-up or death from any cause. At the median follow-up of 10.3 months (range 0.8-142.9 months), 58 patients (92.1%) had progressed and 58 (92.1%) had died. Detailed characteristics of the study population are shown in Table 6.

Plasma Isolation
Peripheral blood samples (12 mL) were collected from all enrolled participants. Samples were collected in Vacutainer ® EDTA Blood Collection Tubes (BD Biosciences, Franklin Lakes, NJ, USA) in the morning on day 0 or day 1 before the first and third doses of chemotherapy. Patient blood samples were centrifuged at 1000× g for 10 min at room temperature within 2 h of venipuncture. To avoid cellular contamination, plasma was carefully harvested and centrifuged again at 1000× g for 10 min at room temperature. The cell-free plasma samples were aliquoted and then cryopreserved at −80 • C. Each sample was thawed only once, immediately before use, for the detection of selected laboratory parameters in the Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Faculty of Medicine, Comenius University in Bratislava, Slovakia.

Selected Laboratory Parameters as a Subject of Interest
The following laboratory parameters were determined in all enrolled patients before systemic treatment initiation and again after two courses of chemotherapy:

Measurement of Lipids
Peripheral blood (6 mL) for the determination of lipids was collected into Vacutainer ® SST TM II Advance (BD Biosciences, Franklin Lakes, NJ, USA) from mUC patients in the morning on day 0 or day 1 before chemotherapy initiation and, thereafter, on day 0 or day 1 before the third course of the same treatment. The samples were processed immediately. TGs, total cholesterol, and HDL-cholesterol were determined by photometry on the Attelica ® chemistry analyzer.
TGs were converted into glycerol and fatty acids by the action of lipase. Glycerol was subsequently converted by glycerol kinase into glycerol-3-phosphate and further by glycerol-3-phosphate oxidase into hydrogen peroxide. A colored complex was formed from hydrogen peroxide, 4-aminophenazone, and 4-chlorophenol due to the catalytic effect of peroxidase. The absorbance of the complex was measured as a reaction with an end point at 505/694 nm.
Cholesterol esters were hydrolyzed by cholesterol esterase to cholesterol and free fatty acids. Cholesterol was converted to cholest-4-en-3-one in the presence of oxygen by the action of cholesterol oxidase to form hydrogen peroxide. A colored complex was formed from hydrogen peroxide, 4-aminophenazone, and phenol due to the catalytic effect of peroxidase. The absorbance of the complex was measured as a reaction with an end point at 505/694 nm.
The test to determine HDL-cholesterol consisted of two different reactions. The first was the elimination of chylomicrons, VLDL-cholesterol, and LDL-cholesterol via cholesterol esterase and cholesterol oxidase. The activity of catalase removed the peroxide produced by the oxidase. The second was a specific measurement of HDL-cholesterol after release by the action of surfactant in the 2 D-HDL reagent. The catalase from step 1 was inhibited by sodium azide in the 2 D-HDL reagent. The intensity of the quinonimine coloration produced in the Trinder reaction was directly proportional to the concentration of total cholesterol measured at 596/694 nm.
VLDL-cholesterol was calculated as TGs/2.2, LDL-cholesterol as total cholesterol minus VLDL-cholesterol and HDL-cholesterol, and the atherogenic index of plasma as log 10 (triglyceride/HDL-cholesterol).

Coenzyme Q 10 and Antioxidants Measurement
Concentrations of CoQ 10-TOTAL (ubiquinone + ubiquinol) and lipophilic vitamins (α-tocopherol, γ-tocopherol, β-carotene) in plasma were determined simultaneously by a modified HPLC method with spectrophotometric detection [33,34]. The oxidation of ubiquinol to ubiquinone was performed with 1,4-benzoquinone before analysis [35]. Plasma samples (500 µL) were extracted by a mixture of hexane/ethanol (5/2 v/v). The tubes were shaken for 5 min and centrifuged at 1000× g for 5 min. The hexane layer was separated and the extraction procedure was repeated with 1 ml of the extracted mixture. Collected organic layers were evaporated under nitrogen at 50 • C. The residues were taken up in 99.9% ethanol and injected into a reverse-phase HPLC column. Elution was performed with methanol/acetonitrile/ethanol (6/2/2 v/v/v). The concentration of CoQ 10-TOTAL was detected with a UV detector at 275 nm, tocopherols at 295 nm, and β-carotene at 450 nm, using external standards. Data were collected and processed with a chromatographic station. Concentrations of analyzed substances were calculated in µmol/L.

TAS and TBARS Measurements
TAS in plasma was determined using the Randox Total Antioxidant Status kit with colorimetric detection at 600 nm. Concentrations were calculated in mmol/L. TBARS were estimated in plasma after reaction with thiobarbituric acid (TBA), quantified spectrophotometrically at 532 nm and expressed in µmol/L [36].

Statistical Analysis
Data were summarized by frequency for categorical variables and by median ± standard deviation and range for continuous variables. p values for categorical variables were calculated using χ 2 or Fisher's exact test and for continuous variables the T-test was used for normally distributed values and the Wilcoxon-Mann-Whitney test was used for non-normally distributed values. The subgroup analyses were accomplished using repeated-measure analysis of variance. The value of statistical significance was set to 0.05. All statistical analyses were performed using NCSS 2022 statistical software, Kaysville, UT, USA [37].

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.