Carnitine Levels Decrease during Oxaliplatin Infusion: A Pilot Study Open Access

Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting side effect of chemotherapy, which might lead to dose reduction or even discontinuation of treatment, thereby negatively affecting chemotherapy efficacy and survival [1, 2]. Moreover, CIPN is associated with several comorbidities such as depression and sleeping disorders and a decreased quality of life among cancer survivors even years after finishing treatment [3, 4]. The development of CIPN depends on several factors including (cumulative) dose, type of anti-neoplastic drug used, duration of administration of the chemotherapy and neuropathy at onset of treatment [2, 5]. Especially platinum compounds, taxanes, and vinca alkaloids are known for their high neurotoxicity [1, 6]. Up to 90% of patients receiving oxaliplatin develop an acute form of CIPN [2]. Approximately 30%–55% of patients treated with chemotherapy develop a chronic form of CIPN, which arises after 3–6 months of therapy and might be permanent [7]. Patients with a chronic form of CIPN predominantly have a sensory neuropathy characterized by glove- and stocking patterned pain, tingling, and/or numbness. Besides, patients could experience motor and autonomic complaints which include loss of strength, difficulty in walking, low blood pressure and constipation [6, 7].

The pathophysiological mechanisms in which anti-neoplastic drugs lead to CIPN are not fully understood yet, but mitochondrial injury and dysfunction seem to play a central role [8]. In short, previous in vivo and in vitro studies showed that oxaliplatin is mitotoxic and induces mitochondrial damage via various pathways [9, 10]. These pathways include damage to the mitochondrial electron transport chain (mETC), resulting in less production of ATP, induction of a higher production of reactive oxygen species, causing a higher amount of oxidative stress and enhancement of mitochondrial apoptosis [8]. Since mitochondria are essential for the generation of energy especially in the axons of nerve cells, mitotoxicity has been linked to the development of CIPN [8].

Carnitine plays an important role in maintaining mitochondrial energy metabolism [11] and it has been suggested as a means of prevention or treatment of CIPN [12]. Levocarnitine, or l-carnitine, is the biological active form of carnitine, which can mainly be found intracellular in muscular tissue and a small amount can be found in plasma. Carnitine is present in our body in two forms: as free l-carnitine and as acylcarnitine esters. Acylcarnitine esters contain carbon chains of various lengths, with acetylcarnitine being the most predominant acylcarnitine. The concentration of carnitine and its esters is maintained between 20 and 50 µmol/L, the acylcarnitine proportion to free carnitine (AC/FC ratio) in plasma remains below 0.4 under homeostatic conditions [11, 13]. The kidneys play an important role in maintaining this homeostasis of the plasma levels of carnitine in our body [14]. Over 90% of l-carnitine is reabsorbed in renal tubules [15]. The AC/FC ratio in urine should remain below 2.0 under normal circumstances [16].

Carnitine is important for mitochondrial metabolism via the carnitine-dependent transport system used for β-oxidation [17]. A deficiency in carnitine may result in a disturbed mitochondrial metabolism, which could be attributed to the development of CIPN. Several in vitro experiments suggested that l-carnitine supplementation might prevent mitochondrial dysfunction via enhancement of pathways involved in mitochondrial energy metabolism [18, 19]. Little is known of carnitine levels in cancer patients and the effect of chemotherapy on carnitine levels. Chemotherapeutic agents with platinum compounds, including cisplatin and carboplatin, have been associated with increased renal excretion and decreased renal absorption of carnitine in cancer patients [20‒22]. Some types of chemotherapy have been able to increase renal carnitine excretion up to ten times [21]. Based on the results of these previous studies [20‒22], it was thought that platinum-based chemotherapeutics such as oxaliplatin induce carnitine deficiency by increasing renal carnitine excretion. This carnitine depletion will be reflected in decreased plasma carnitine levels and increased urinary carnitine levels [23] and might eventually contribute to mitotoxicity and development of CIPN.

Until now, no effective treatment has been available for patients with CIPN. Moreover, no preventive strategy has been developed yet to prevent the occurrence of CIPN in patients receiving oxaliplatin-based chemotherapy [24, 25]. Clinical intervention studies toward the effects of carnitine supplementation during chemotherapy infusion show contradictory results [1, 12, 26]. This may be due to differences in timing and method of carnitine administration. To be able to set up an intervention study using carnitine supplementation as a preventive strategy for CIPN development in patients receiving oxaliplatin, it is crucial to first achieve better understanding of the effects of oxaliplatin on the course of carnitine levels.

The aim of this study was to investigate whether oxaliplatin-based chemotherapy administration affects blood and urinary levels of carnitine during and directly after oxaliplatin administration. It was hypothesized that both serum and urinary levels of carnitine will change during chemotherapy infusion as a result of increased renal excretion.

This pilot study was a single-center prospective observational study, which was carried out in the outpatient oncology clinic of the Maxima Medical Center (MMC) in Eindhoven and Veldhoven, The Netherlands. From February 2019 through December 2019, all patients planned to start with oxaliplatin-based chemotherapy at MMC were eligible for participation in this pilot study. Eligibility criteria to participate in this pilot study included (1) written informed consent, (2) age ≥18, (3) well understanding of the Dutch language and (4) patient will start treatment with oxaliplatin-based chemotherapy after inclusion. Exclusion criteria included (1) known primary carnitine deficiency (congenital), (2) treatment with hemodialysis or peritoneal dialysis, (3) known epilepsy, (4) use of carnitine supplements currently or in the past 3 months, (5) pre-existent neuropathy, (6) previous treatment with neurotoxic chemotherapy and/or (7) participation in an intervention study on CIPN. Sample size was not calculated by means of a power calculation since this pilot study was set up as preparation for a larger intervention study in the future. However, since previous studies were able to show clinically relevant changes in carnitine levels during treatment with chemotherapy in relatively small study populations (n = 5–11) [20‒23], we decided to observe 10 patients, assuming this would be sufficient to see significant changes in both plasma and urine carnitine levels.

Ethical approval for the study was obtained from the certified Medical Ethics Committee of Medical Research Ethics Committees United. The guidelines on Good Clinical Practices were fully applied, complying with current Dutch legislation on clinical research in humans and with the Declaration of Helsinki. All patients provided written informed consent.

Patients eligible for inclusion were identified by the specialized oncology nurse, then asked to participate before the start of their first cycle of oxaliplatin-based chemotherapy. In case consent was given, patients were asked to fill in a baseline questionnaire. Thereafter, blood and urine samples were collected before, during and after administration of the first cycle of oxaliplatin. Oxaliplatin was administered intravenously and took 2 h to complete. One blood and urine sample was taken before the infusion of oxaliplatin (baseline measurement), three blood samples were collected during infusion of oxaliplatin (at T-minutes since baseline: T5, T60, and T120) and one blood and urine sample was taken 1 h after infusion of oxaliplatin (T180) (Fig. 1). Follow-up ended 1 h after completion of the first cycle of oxaliplatin. Both blood and urine samples were stored on ice at −20°C. After the collection of samples of all participants, the samples were sent to the laboratory for Genetic Metabolic Diseases of Amsterdam University Medical Center for determination of the full carnitine spectrum. Moreover, electronic patient files were used to obtain tumor and treatment-related information.

Primary endpoint of this study was the plasma and urinary concentrations of full carnitine spectrum before, during and after administration of oxaliplatin-based chemotherapy. Full carnitine spectrum consisted of free carnitine (C0), acetylcarnitine, and full acylcarnitine ester spectrum (C2–C18). Within the acylcarnitine spectrum, carnitine esters were categorized by length, based on the number of carbon atoms. This resulted in a division into three groups, namely, short (2–5 carbon atoms), medium (6–12 carbon atoms), and long (13–18 carbon atoms) chain carnitine esters. See Table 1 for a detailed overview of carnitine ester spectrum determination.

The following baseline characteristics were obtained from our questionnaire: date of birth, gender, height and body weight to calculate BMI, information about dietary intake (full diet, vegetarian, vegan), use of dietary supplements in the past 3 months, nicotine consumption, alcohol consumption, comorbidities, type of cancer, cancer stadium (TNM classification), scheme of chemotherapy received, and history of previous chemotherapy (type, indication, duration of administration). Moreover, patients were screened for the following comorbidities: cardiovascular diseases, stroke, obstructive lung diseases, connective tissue disorders, paralysis, kidney disorders, disorders of the gastro-intestinal tract, liver disorders, diabetes mellitus, HIV/AIDS, and malignancy treated (not treated with chemotherapy).

All statistical analyses were performed using SPSS statistics (IBM SPSS Statistics for Windows, versions 22.0, released 2013; IBM Copr, Armonk, NY, USA) Descriptive statistics were used to analyze baseline characteristics of the included patients. Non-parametrical tests (e.g., Wilcoxon signed-rank test) were used to analyze the primary endpoints of this pilot study. All tests were two-sided, considered statistically significant if p < 0.05.

A total of 17 patients were eligible for study inclusion, of which 10 patients participated (Fig. 2). Patients did not participate due to logistical reasons, failed sample collection, or declined to participate. Five blood samples and two urine samples were obtained from each participant. Two urine samples at baseline were missing due to lack of urge to urinate. Baseline characteristics are summarized in Table 2.

The study included both men and women, aged between 46 and 71. All patients were diagnosed with cancer of the gastro-intestinal tract and treated with oxaliplatin combined with capecitabine (CAPOX), oxaliplatin combined with 5-fluorouracil and folic acid (FOLFOX) or oxaliplatin combined with 5-fluorouracil, folic acid, docetaxel, and dexamethasone (FLOT). These treatment schedules all start with oxaliplatin as the first drug to be administered.

The course of carnitine concentrations in plasma showed a decrease in total carnitine during oxaliplatin infusion compared to baseline (measuring moment 1). This decrease was seen in free carnitine, total acylcarnitine spectrum, and acetylcarnitine (Fig. 3).

Total plasma carnitine was significantly lower at the end of chemotherapy infusion compared to total plasma carnitine at baseline (Table 3). This decrease in carnitine level at the end of chemotherapy infusion was also seen in free carnitine levels, total carnitine ester spectrum levels and acetylcarnitine levels.

However, 1 h after finishing infusion of chemotherapy, this decrease was not significant anymore (Table 4). The AC/FC ratio was 0.2 before the chemotherapy infusion. Directly after infusion of oxaliplatin, the AC/FC ratio decreased significantly to 0.15 (p = 0.005), indicating a relatively greater decrease in acylcarnitines compared to the decrease in free carnitine. When classifying carnitine esters in plasma by length, a significant decrease in carnitine esters was seen in all three groups (short-, medium-, and long-chain carnitine esters) directly after infusion of chemotherapy compared to baseline (Fig. 4).

In urine, the total carnitine concentration after chemotherapy infusion was increased as compared to baseline (before chemotherapy administration). However, this increase was not significant (Table 3). The concentration of free carnitine, full acylcarnitine spectrum, and acetylcarnitine in urine seemed to increase after chemotherapy. The AC/FC ratio in urine before chemotherapy infusion was 1.23 and decreased significantly (p = 0.017) to 0.69 after chemotherapy infusion.

This pilot study showed a significant decrease in both free carnitine and full carnitine spectrum in plasma 2 h after start of infusion of oxaliplatin-based chemotherapy. Moreover, a non-significant increase in carnitine concentration in urine during chemotherapy infusion was seen. The results of this study are in line with previous studies showing that chemotherapeutic agents are able to affect carnitine levels in our body. Administration of platinum compounds such as cisplatin and carboplatin lowered total body carnitine levels in cancer patients due to increased renal excretion and inhibited reabsorption of l-carnitine [11, 20, 22, 27]. While our study showed that plasma concentration of l-carnitine significantly decreased during administration of oxaliplatin, treatment with other platinum compounds showed relatively stable plasma levels of l-carnitine. These stable plasma concentrations may be the result of a decrease in the intracellular l-carnitine pool or a shift in the ratio between free carnitine and different acylcarnitine esters. Together, the altered plasma and urinary concentrations of l-carnitine support our hypothesis that oxaliplatin causes increased renal excretion of carnitine, thereby lowering blood-carnitine levels and increasing urinary carnitine levels.

Although the absolute changes in both plasma and urinary concentrations of l-carnitine were small, these changes might be of clinical relevance, especially when accumulated over time. A previous study showed that increased renal excretion might continue for days after completion of chemotherapy infusion [23], indicating that plasma l-carnitine levels might further decrease after our follow-up period has ended. Moreover, studies showed that patients can lose up to 10% of their total body carnitine during one cycle of chemotherapy [20, 21]. Repeated cycles of chemotherapy will increase this percentage of lost l-carnitine. The cumulative loss of l-carnitine resulting from multiple treatments with oxaliplatin might eventually result in a deficiency of carnitine. Therefore, the small but significant decrease in plasma concentrations of l-carnitine after only one cycle of chemotherapy may mark the beginning of the development of a carnitine deficiency which was triggered by oxaliplatin administration. Furthermore, l-carnitine homeostasis is necessary to preserve mitochondria functioning and fatty acid metabolism within the mitochondria [11, 13]. Even a slight disruption of these homeostatic conditions by decreased plasma levels of l-carnitine might result in mitotoxicity. Previous studies showed that mitotoxicity is a major cause of CIPN [8, 28]. Thus, since l-carnitine levels are normally kept in homeostasis, even small differences in l-carnitine concentration or composition of the acylcarnitine spectrum might contribute to the development of CIPN, especially when accumulated over time.

Several (methodological) limitations of this study should be addressed. First, this pilot study lacked a control group and had a small sample size. Since course of carnitine levels in the same patient was of our primary interest, and carnitine normally remains stable within known limits, a control group was not necessary for this pilot study. As mentioned above, previous studies were able to show significant results with small sample sizes. Our study was also able to yield significant results despite the small sample size. However, our study might still have lacked power, especially in the urine sample testing. Two missing urine samples at baseline might have negatively affected power, causing the alterations in urinary carnitine levels to not be significantly changed compared to baseline. Future studies with larger sample sizes are necessary to confirm these findings. Furthermore, the degree of dilution of urine might have affected the urine results, for which this study could not correct. This might have resulted in a greater spreading of carnitine measurements, which also further impedes yielding of significant results.

Another limitation of this study is the short follow-up period and lack of multiple measurements after completion of chemotherapy infusion. As previously mentioned, other studies showed increased renal excretion after administration of platinum-based chemotherapy. Haschke et al. [23] showed increased renal excretion of carnitine reaches its maximum 1 or 2 days after chemotherapy administration and renal excretion remains elevated for several days after chemotherapy infusion. Therefore, multiple measurements in the days after completion of oxaliplatin infusion would be preferable to being able to investigate the effect of peak and prolonged renal excretion on carnitine levels. Moreover, a longer follow-up period would make it possible to investigate whether carnitine levels in plasma restore between two cycles of chemotherapy. Since our aim was to study the acute effects of oxaliplatin administration on carnitine levels, the chosen follow-up time was sufficient to be able to answer the research question of this pilot study. However, a longer follow-up, assessing carnitine levels over multiple chemotherapy cycles, would be valuable to investigate cumulative carnitine depletion and potential correlation with the onset of CIPN symptoms. Also, systematic collection of clinical toxicity data, such as symptoms of CIPN, was not part of the study protocol. This is a limitation of the current study and highlights the need for future research to include both biochemical and clinical endpoints to better understand the relationship between carnitine dynamics and chemotherapy-induced peripheral neuropathy.

We studied l-carnitine levels in plasma and urine, while most of our body carnitine can be found intracellular in muscular tissue. However, determination of carnitine in muscle tissue requires muscle biopsies, which carry a high patient burden. Moreover, we expected changes in muscular carnitine levels to occur not immediately, but hours to days after oxaliplatin administration [13]. Since endogenous carnitine in plasma and tissue carnitine are in strict homeostasis, alterations in plasma carnitine level will cause tissue carnitine levels to change over time to maintain homeostasis [13]. Although measures of l-carnitine in muscular tissue would give an optimal overview of total body carnitine status, measurements in plasma are a suitable patient-friendly alternative to study the effects of chemotherapy on total body carnitine levels.

In conclusion, administration of oxaliplatin causes decreased total plasma carnitine levels, and alteration of both the plasma and urinary profile of l-carnitine after one cycle of chemotherapy. Although the absolute loss of total body l-carnitine is small, even these small alterations require adaptation of the carnitine homeostasis and could, when accumulated over time, possibly contribute to the development of CIPN. Since this study helped clarify course of l-carnitine and full acylcarnitine spectrum during chemotherapy administration, this could be helpful in the determination of timing and dose of l-carnitine supplementation for future intervention studies. It would be interesting to investigate if the acute loss of l-carnitine in plasma can be compensated by supplementation of l-carnitine. Besides, future studies should focus on whether carnitine levels recover between cycles or if a chemotherapy-induced carnitine deficiency will occur after repeated cycles of oxaliplatin. A better understanding of l-carnitine homeostasis during oxaliplatin administration is necessary to eventually investigate the role of carnitine as a preventive strategy for the development of CIPN.

The authors would like to thank all patients for their participation in this study. The authors give special thanks to R.B. Berendsen, who was willing to function as advisor for the team of investigators. In addition, we want to thank Dr. F.M. Vaz for his advice and critical notes upon this study.

Ethical approval for the study was obtained from the certified Medical Ethics Committee of Medical Research Ethics Committees United, Nieuwegein, The Netherlands, with approval No. NL65037.015.18. The guidelines on Good Clinical Practices were fully applied, complying with current Dutch legislation on clinical research in humans and with the Declaration of Helsinki. All patients provided written informed consent.

The authors declare that they have no conflict of interest.

The present research was funded by AlfaSigma Nederland BV, Utrecht, The Netherlands.

Lianne P.W. van Geffen and Veerle C.M. Geurts: design, performed research, analyzed data, and writing. Floortje Mols and Gerard Vreugdenhil: design, supervision, review and editing, revision.

The data that support the findings of this study are not publicly available due to privacy considerations but are available via the first author (L.P.W.G.) upon reasonable request.