New possibilities of application of differential scanning calorimetry—new clinical diagnostic methods on the horizon?

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Differential scanning calorimetry (DSC) is the most often used method in thermal analysis mainly applied in the research field, but according to the results, its clinical application emerges too. With this method, we can analyze the structural change of biological material so we can measure the effects of drugs and we can quantify and characterize changes in body. Because of this, it is possible to conclude short- and long-term effects in a predictive way. This paper is a comment on paper published by Farkas et al.¹

Our team were examined the effects of cyclophosphamide—which is a widely used cytotoxic drug and appears on the most important drugs' list made by WHO—on guinea pigs equivalent to human protocol in experimental conditions. Cyclophosphamide is a very effective immunosuppressive drug—used primarily in severe immunological diseases associated with organ involvement such as scleroderma, vasculitis, SLE and also used in organ transplantations. In addition, in high dose and often combined with other chemotherapeutic drugs, it is a most relevant antineoplastic drug that can damage slowly and rapidly segmented cells at the same time. It is important to know that the metabolism of cyclophosphamide is partly genetically determined and partly influenced by comorbidities and incidental treatment. Therefore, in the case of tumors and well-known autoimmune diseases with different pharmacokinetics, it shows a large individual variability and may change with time. Besides its beneficial effects, cyclophosphamides may have got severe life-threatening side effects and complications because of the actual plasma level and high cumulative dosage. Cyclophosphamide's carcinogenic effects are known. Its usage may cause acute myeloid leukemia (AML) and urinary bladder cancer. Severe side effects are bleeding cystitis, thromboembolism, infertility, and teratogenic effect.

In the first step of our experiment, we examined the effects of cyclophosphamide on guinea pigs' nerve-muscle complex with oncological indication by using cyclophosphamide dosage and protocol based on body mass.¹ The analyses were made by the SETARAM Micro DSC-II calorimeter. According to our results, we could show a significant, dose-dependent difference between thermal parameters (T_m = denaturation temperature, ΔH = calorimetric enthalpy, not marked) of untreated and treated samples, which proved that cyclophosphamide has got a peripheral and in smaller degree of cases muscle damage effect (see Fig. 1). In the case of muscle, we can join these changes to alteration in the ATPase function of myosin head (first peaks in Fig. 1B) as well as to change in the structure of actin filaments (third peaks in Fig. 1B). As a second step with the same method and cyclophosphamide dosage, we examined the heart muscle and also detected the treatment-caused alterations with DSC.² In third step to keep in mind the further clinical application, we perform the experiment with unchanged parameters in blood plasma too and our results clearly demonstrate the detection of dosage-dependent changes on plasma.³

Figure 1. Denaturation curves of Guinea pig n. ischiadicus (A) and m. gastrocnemius (B). Downward deflections are endotherm processes. Symbols: i-injection, d-days between the injection or exit, e-exit (see ref. 1, no permission is required).

By collectively evaluating the performed studies, a clear correlation can be observed in the results obtained by different materials. Alterations in different materials caused by using different cyclophosphamide dosages and not uniform treatment length produced well-correlated and clearly detected changes with DSC. For this reason, if we can detect the drug-induced changes for example in blood plasma, we can conclude effects in other areas too. Based on our results, we found detectable and partway quantified alterations with DSC on blood plasma components; therefore, it can be used in clinical routine.

Most of the drugs used in therapy such as cyclophosphamide show varying plasma concentration in the function of individual circumstances and clinical conditions beside the standard applied dose. Considering that in the case of numerous drugs there is a small difference between therapy and toxic side effects, it could be useful to apply this relative simple method for monitoring. The direct determination of drug levels in most of the cases is difficult and expensive—the cyclophosphamide level of plasma can be monitoring by liquid chromatography—mass spectrometry that is an effective but a circumstantial method. In contrast to this, the testing of blood plasma using DSC is relative simple and cheap. If we could define quantifiable relationships between denaturation temperatures, calorimetric enthalpy differences, and thermogram modifications detected by DSC, then we can get information about induced effects in other areas of the body. In long-term treatments, incidental severe results and side effects caused by cumulative dose may become predictive with this method.

Taking a blood sample is minimal invasive—in severe diseases, it is a part of daily routine—therefore acceptable for the patients. The evaluation of sample could be partially automated, so we can get a relative fast result and make further therapy decision.

In the last years, numerous studies had been published for plasma examination with DSC that primarily focused on tumors detection, follow ups, and monitoring based on thermogram changes of plasma and proteins.^4,5 According to our results, thermogram is very sensitive and informative and highlights the opportunities of DSC's clinical application as a complementary diagnostic tool.

Our research differs from studies that we have mentioned before, because we not only examined primary pathological changes and diseases, but also focused on the prediction of treatment effectiveness of drugs used by us and expected side effects and complications. If we can manage to predict the harmful effects for patients that are arising from too high blood plasma concentration and cumulative dose with this method, we could prevent them by decreasing the dose or changing to other drug. This is beneficial for the patient and treatment too.

This new area means a new challenge. In addition to that, we could determine the exact drug dosage with this method; it is necessary to safely quantify the detectable changes by considering the modifications caused by disease. This is easily made with a basic test before therapy, but it is important not to ignore the changes due to the progression of patient's disease and appearance of associated diseases.