Journal of Thermal Analysis and Calorimetry

, Volume 113, Issue 1, pp 259–264

Differential scanning calorimetry (DSC) of blood serum in chronic obstructive pulmonary disease (COPD)

A new diagnostic tool ahead?

Authors

  • Zsuzsanna Szalai
    • Department of PulmonologyPetz Aladár Hospital
    • Department of PulmonologyKarolina Hospital
  • Tamás F. Molnár
    • Thoracic Surgery Unit, Department of SurgeryPetz Aladár Teaching Hospital
    • Faculty of Medicine, Institute of BiophysicsUniversity of Pécs
Article

DOI: 10.1007/s10973-013-2999-1

Cite this article as:
Szalai, Z., Molnár, T.F. & Lőrinczy, D. J Therm Anal Calorim (2013) 113: 259. doi:10.1007/s10973-013-2999-1

Abstract

Chronic obstructive pulmonary disease (COPD) is a major global health challenge with a gloom perspective of being one of the big three cause of death by 2020. No reliable/reproducible biomarker has been identified so far to match the clinically-based staging system (GOLD). Blood samples of 30 subjects divided into 6 groups (no-COPD/-smoker, no-COPD/non-smoker, COPD I, COPD II, COPD III, COPD IV) with 5 patients in each were tested by differential scanning calorimetry. There is a clear 15.4 % difference between the heat flow maxima measured when no-COPD subjects were compared in accordance to their smoking/non-smoking status. Odds ratio of different heat flow in actively smoking COPD patients in stage IV and stage I was 1.61. A reverse tendency is detected in the relevant non-smoking COPD groups. The differences are inconsistent in intermediate stages (COPD II and III). DSC seems to be an applicable and objective method for monitoring nicotine abuse. There is a chance to detect specific typology of thermokinetic patterns in the two extremes of COPD (I vs. IV). Further studies with increased sample size are needed to allow calculations on specificity/sensitivity/positive and negative predictive value of enthalpies and heat flow maximums. The first clinically relevant blood-based COPD marker on the intravascular side of the alveo-capillary screen is demonstrated by our pilot study.

Keywords

Differential scanning calorimetry (DSC)Chronic obstructive pulmonary disease (COPD)COPD severityGlobal initiative for chronic obstructive lung disease (GOLD)

Introduction

Chronic obstructive pulmonary disease (COPD) is a major global health challenge leading to serious economical and individual consequences. By definition it is a preventable and treatable respiratory disease with some significant extrapulmonary effects, associated with an abnormal inflammatory response of the lung to noxious particles (such as smoking) or gases. The pulmonary component is characterized by progressive and not fully reversible airflow limitation. [1]. According to recent WHO/World Bank reports (GOLD Reports), COPD is the fourth leading cause of chronic morbidity and mortality in the United States at the time being and is projected to rank third cause of death worldwide by 2020 [2]. Irreversible and progressive destruction of the lung parenchyma resulting in inability to provide sufficient gas exchange via ventilation/perfusion is the very essence of the clinical picture [3]. There are wide individual variations in the pace of the complex inflammatory process of lung parenchyma. Intensive pharmaceutical research on pathways is underway to slow down or even temporary halt the injury cascade and to facilitate reparative processes. While understanding of COPD is still far from complete, there is a consensus on terms of a severity score guided treatment strategy providing platforms for large volume clinical trials. Four clearly defined stages of COPD are distinguished for comparative analysis of phenotypes of the disease [4]. While, these guideline-friendly categories are easy to use in daily clinical practice, their pathophysiological background is less than well- established [5].

A major impediment in clinical research has been the lack of lung-specific biomarkers applicable as an intermediate end point for short-term trials. Biomarkers are expected to be biological plausible reflecting to the pathogenesis of COPD and to match with outcome (hospitalisation, mortality). The recorded intervention should be monitorable to change the target outcome of interest [7]. Biomarkers investigated in COPD so far have failed to fulfil these criteria as no specific and stage- dependent values are established [6]. A long list of mediums like induced sputa, BAL fluid, bronchial biopsy specimens, and exhaled breath condensates have been investigated without definitive result. Major methodological shortcomings, including cost-benefit issues, invasivity, unsatisfactory reproducibility and problems with measurement standardization blocked routine clinical application [8]. With the paradigm shift of COPD seen it as a systemic disease clinical research has been focused on blood specimens [9] rather than tissue samples.

Serum or plasma biomarkers such as high sensitive C-reactive protein (hs-CRP), interleukin-6, pulmonary activation-regulated chemokine [10] and inhibitors of plasminogen activators relate to lung function and important outcome data such as exacerbations, morbidity, and mortality [11]. Elevated blood hs-CRP levels are associated with major outcomes including lung function reduction, hospitalisation, mortality [9, 12, 13] and increased risk of exacerbations [14]. Contradictory reports [15] on moderate-to-severe COPD are challenging predictive value of CRP levels. Physiologic measurements (FEV1, arterial oxygen tension, inspiratory capacity/total lung capacity ratio and 6-min walk test) are proven to be related to mortality, as previously reported [810, 15]. Comorbidities and medicines/drugs are also influencing biomarkers [16, 17]. Functional tests are scanty and mainly limited to the investigation in blood rheology in COPD [18, 19].

Differential scanning calorimetry (DSC) of blood serum shed a light on correlation between enthalpy and biological behaviour of normal and abnormal tissues [2022]. The DSC method is monitoring protein folding and unfolding reactions accompanied by heat effects. The heat of unfolding measured at a constant pressure represents the enthalpy of the process. Direct measurement of the heat of unfolding or phase transition by DSC is proven an applicable method for determining thermodynamic properties of biologically active macromolecules [23, 24]. Possible connection between chronic obstructive pulmonary disease and blood serum denaturation transition was investigated recently [25]. Our hypothesis was that these structural differences in the blood sera of patients are COPD stage-dependent not only reflecting to severity of disease but mirroring effect of smoking, also.

Patients and methods

Human blood samples

Having obtained the permission of the Regional Ethic Committee of University of Pécs, Hungary (4112/2011) all consecutive consenting COPD patients of Pulmonology Department, Karolina Hospital Mosonmagyarovar, Hungary were enrolled, till the relevant group sample sizes have been completed. The study was undertaken following a one patient-one test-performed once measurement concept. During the patients’ programmed routine check up, an extra 5 ml blood sample was taken for DSC investigations. Interview, physical examination, pulse oximetry and lung function test has completed the session. Groups were formed in age, gender, smoking status and socio-economic group to be matched. Altogether six groups were formed either of them originally consisting of five patients. Two reference groups provided healthy controls/baseline values. One group consisted of active smokers (group 1), while non-smokers formed the opposing arm (group 2). Four further group of patients (groups 3–6) were formed in accordance with their GOLD severity stages of COPD I–IV. Table 1 contains the demographics and sex distribution of the subject groups.
Table 1

Age and sex distribution of subjects (n = 30) among different severity and control groups

Group of patients

Male/female

Age mean/range

FEV1 mean/range

Control groups

 Group 1 smokers

3:2

30.1 SD 4.8 (19–42)

 

 Group 2 non-smokers

2:3

41.1 SD 2.3 (37–53)

 

COPD groups (GOLD)

 Group 3 I (FEV1: >80 %)

2:3

50.8 SD 1.1 (43–54)

85.2 % SD 1.3 (82–94)

 Group 4 II (FEV1: 50–80 %)

2:3

56.8 SD 4.2 (48–70)

67.1 % SD 2.1(61–73)

 Group 5 III (FEV1: 30–49 %)

4:1

62.1 SD 4.7 (51–76)

40.5 % SD 2.2 (30–48)

 Group 6 IV (FEV1: <30 %)

5:0

58.6 SD 1.8 (55–61)

27.6 % SD 1.3 (24–29)

GOLD the global initiative for chronic obstructive lung disease, FEV1 percent of predicted forced expiratory volume in the first 1 s of expiration

DSC investigations

The blood serum samples were coded and the test was blinded at the DSC measurement point. Samples were stored at −20 °C till investigation. The thermal unfolding of the human plasma components were monitored by SETARAM Micro DSC-II calorimeter. All experiments were conducted between 0 and 100 °C. The heating rate was 0.3 K min−1 in all cases. Conventional Hastelloy batch vessels were used during the denaturation experiments with 950 μL sample volume in average. Reference sample contained normal saline (0.09 % NaCl). The sample and reference samples were equilibrated with a precision of ±0.1 mg. There was no need to do any correction from the point of view of heat capacity between sample and reference samples. The repeated scan of denatured sample was used as baseline reference, which was subtracted from the original DSC curve. Calorimetric enthalpy was calculated from the area under the heat absorption curve by two-point setting SETARAM peak integration.

Results

Table 2 summarizes the thermal denaturation parameters of blood serum of patients of the different groups (groups 1–6). Measurement failures resulted in drop out of one patient from group 1, 4, 5 each.
Table 2

Thermal denaturation parameters of blood serum proteins obtained from different patient groups (with or without COPD, no-smokers, smokers and those who gave up the smoking)

Groups

Patients

# of patients

Thermal parameters

Patients

# of patients

Thermal parameters

Tm/°C

ΔH/J g−1

Tm/°C

ΔH/J g−1

1.

Smokerno-COPD

4

63.60 ± 0.2

1.29 ± 0.06

    

2.

Non-smoker no-COPD

5

63.30 ± 0.2

1.28 ± 0.06

    

3.

Smoker COPD-1

5

63.44 ± 0.2

1.34 ± 0.07

Gave up smk. COPD-1

4.

Smoker COPD-2

2

64.11

1.41

Gave up smk. COPD-2

2

63.78

1.33

5.

Smoker COPD-3

2

63.16

1.32

Gave up smk. COPD-3

2

64.38

1.50

6

Smoker COPD-4

2

63.28

1.62

Gave up smk. COPD-4

3

63.70

1.41

 

Total

15

   

12

  

The data are in mean ± SD (SD is missing in cases when number of samples were smaller than four)

Figure 1 clearly demonstrates the thermal consequence of smoking in the control group (COPD free patients: group 1 vs. 2). There is a clear 15.4 % difference between the heat flow maxima measured when no-COPD subjects were compared in accordance to their smoking/non-smoking status (n 4 vs. 5). The DSC scans serve as a fingerprint for the progression of lung disease in case of smoking COPD patients as it is exhibited in Fig. 2. The difference between the heat flow maxima in the most advanced stage COPD IV and the early stage COPD I is a striking 60.9 % representing a possible odds ratio of 1.61. The differences between the intermediate stages, i.e. COPD II and III are not so impressive, but quite consistent. The decrease of melting points can refer for the decreased thermal stability while the increased calorimetric enthalpy could mean the decreased flexibility (the structure of influenced serum proteins becomes more rigid) as the disease advances.
https://static-content.springer.com/image/art%3A10.1007%2Fs10973-013-2999-1/MediaObjects/10973_2013_2999_Fig1_HTML.gif
Fig. 1

DSC scans of blood serum of healthy smoking and no smoking control subjects (no-COPD)

https://static-content.springer.com/image/art%3A10.1007%2Fs10973-013-2999-1/MediaObjects/10973_2013_2999_Fig2_HTML.gif
Fig. 2

Thermal denaturation of blood serum of smoker COPD patients: COPD I–IV (GOLD)

Difference between thermal parameters of non-smoking COPD IV and non-COPD (COPD I equivalent) shows the reverse pattern as their smoking counterparts. However, the differential value between the two extremes are the same in both scenarios as seen on Fig. 3. The calorimetric enthalpy seems to be consistent with the clinical phases in the healthy/very early and in the most serious cases. It seems to be obvious, that in the most advanced stage of the disease the enthalpy curve patterns are diagonally opposite in the two groups: i.e. active smokers versus non-smokers, respectively (see Table 1; Figs. 2, 3). Intermediate stage COPD blood samples were less consistent in their enthalpy-disease severity relations.
https://static-content.springer.com/image/art%3A10.1007%2Fs10973-013-2999-1/MediaObjects/10973_2013_2999_Fig3_HTML.gif
Fig. 3

Unfolding of blood serum proteins of non-smoker COPD patients: COPD I–IV (GOLD)

Discussion

The original hypothesis, that COPD stage-specific enthalpy patterns are to be detected was overwritten by the obvious difference in thermic features of blood samples of smoking and non-smoking subjects. This basic difference led to a change in interpretation of our results and a consequent reduction in sample sizes. The mild but consequent increase in melting temperature and calorimetric enthalpy in cases of COPD free smokers is a sign of global structural change in serum proteins. Due to the small number of patients it would be too early to draw statistically based conclusions, but at this stage a watershed seem to emerge with regard to the objective detection of the progression of the disease.

Our data on blood samples of healthy subjects and COPD sufferers, smokers and ex-smokers alike, are offering more than one explanation. The results are proving, that calorimetry is an objective method for calibrating nicotine abuse (smoking).

The rather distinctive typology of smoking related heat flow curves and consequent enthalpy patterns (active smokers vs. non-smokers) might reflect to a clinically relevant difference between the biological behaviour of blood sera of the two main entities. (Fig. 1). Nicotin abuse seriously but reversibly affects healthy subjects’ blood sera enthalpy pattern. We do have an objective measuring method for proving a self-destructive behaviour. DSC seems to be an applicable and objective method for monitoring nicotine abuse. Our sample size does not allow yet to calculate specificity/sensitivity/positive and negative predictive value of the method. A significantly increased sample size is definitely needed. The same refers to the the question of differentiating between the four stages of COPD in the individual patient. There is a clear tendency in the enthalpies measured in the different groups (groups 3–6) according to the severity of the clinical picture (Fig. 2). However as the present smoking status (yes/not) has a strong influence on the baseline enthalpy pattern (Table 1) no statistical analysis is allowed due to low sample sizes in the identical subgroups. Behaviour and interaction of proteins [26] in blood serum mixtures investigated by excessive thermal insults is dependent on multifaced factors, like pathology under investigation, mixture and severity of concomitant diseases drugs and diet, nutritional status and age just to mention the leading elements of a long list [27, 28]. While, one of the shortcomings of the present preliminary study is the application of definitely younger control groups, there is a clear difference even among the identical control groups (smoker vs. non-smoker) where enthalpy and heat flow patterns are considered. This difference is less expressed, but is quite consistent all the way along from severity group COPD I–IV. Exactly this basic difference between smokers/non-smokers prevents us to conclude more explicitly when stage- specific enthalpy patterns and heat flow values are sought of. The inavoidable significant differences in ages within the different severity groups (COPD I–IV) is rooted in the natural process by time of the disease under investigation itself. Explanation of the observed COPD stage-connected DSC patterns might include circulation related factors [2931]. Cold-agglutinins, thermally instable inflammatory mediators and alterations in blood viscosity are on the suspects list [32, 33] not only for pathogenesis but also for mortality of COPD patients [34]. Data on smokers and an patients in stage IV COPD are distinctively different from the rest of the examined pool even in this preliminary study.

Conclusions

Blood serum of COPD patient is difficult to analyze by use of thermal methods due to its extremely complex nature. However there seem to exist distinctive patterns detectable by DSC between the different severity stages, a long awaited objectively monitorable feature. DSC technique seems to present a COPD stage-specific method purely on the thermokinetic characteristics of the individual patient. So far pneumonology failed to achieve an objective assessment/staging method other than lung function test in COPD. The first clinically relevant blood-based COPD marker on the intravascular side of the alveo-capillary screen is demonstrated by our pilot study. Large cohort, cross section, repeated measures/same patient dynamic examinations are definitely needed in the close future. These studies are underway.

Acknowledgements

The SETARAM Micro DSC-II was purchased with a grant (CO-272) from the Hungarian Scientific Research Fund (D. Lőrinczy).

Copyright information

© Akadémiai Kiadó, Budapest, Hungary 2013