Introduction

Duchenne muscular dystrophy (DMD) is an X-linked disorder, it is the most common muscular dystrophy in children, present in early childhood and characterized by proximal muscle weakness and calf hypertrophy in affected boys. It occurs 1 in 3500 live male births. This disease is caused by the mutation in dystrophin gene located at Xp21.2 that codes for dystrophin protein, an important structural component of muscle cell [1].

Diagnosis of DMD is carried out on the basis of clinical symptoms (muscle wasting and weakness, difficulty in walking and standing, and frequent fall) and, estimation of CK (Creatine kinase) activity and gene mutation analysis. Most of the children suffering from DMD develop proximal muscle weakness by 5 years of age and are unable to walk by the time they reach 8–12 years of age [1, 2]. Estimation of CK levels in serum is a simple and inexpensive method to detect muscular dystrophies, but this test is not specific since, levels of CK are increased in all types of myopathies [1]. Activity of serum CK in DMD patients can be 50–100 times higher as compared to normal levels even at birth, even before the disease becomes clinically evident [1]. Gene mutation analysis of these patients can be performed using peripheral blood and the diagnosis can be established in most of the cases [3, 4]. Around 30 % DMD cases are reported negative when diagnosed using gene mutation analysis due to point mutation (single base substitution in the gene) in the dystrophin gene [4]. Multiplex PCR is a commonly used method for DMD diagnosis in India, which targets about 18–32 exons of the dystrophin gene to identify complete exons deletions. Multiplex PCR is mostly qualitative or semi-quantitative and serves for the exons in the hot spot region [5]. MLPA (multiplex ligation-dependent probe amplification) has facilitated more reliable and quicker quantitative deletion of the complete dystrophin gene containing 79 exons to study the deletions and duplications. Multiplex PCR method permits the detection of approximately 98 % of deletions, which accounts for 65 % of all mutations [5, 6]. Following multiplex PCR by MLPA increases the percentage of patients with a precise diagnosis to 75 %. MLPA-based diagnostic method is used to diagnose DMD in USA, China and several European countries [6, 7]. Diagnosis using immunohistochemistry is a crucial diagnostic method for cases which are negative gene mutation cases of DMD and exclusion of the congenital muscular dystrophy [8], McLeod syndrome [9, 10], miscellaneous myopathic and neurogenic disorders, such as the autosomal recessive form of limb girdle muscular dystrophy [11], primary alpha- and gamma-sarcoglycanopathies [12, 13], spinal muscular atrophy [14] and in Charcot-Marie-Tooth disease type 1A [15].

Nuclear Magnetic Resonance (NMR) spectroscopy is a versatile technique that can be used in a broad series of discipline. The best known medical applications of NMR are in vivo magnetic resonance imaging and spectroscopy, but in vitro NMR spectroscopy of body fluids such as blood plasma or serum has also been used to diagnose inborn errors of metabolism [16–20]. Various authors have studied different individual blood components to find out any abnormalities that could be specific to DMD cases. Analysis of the biochemical, morphological and biophysical characteristics of red blood cell (RBC) membrane demonstrated an alteration in both myotonic dystrophy and DMD [21]. Higher levels of free fatty acids and ketone bodies were detected in serum of DMD patients [22]. Studies of lipid profile in dystrophic chicken and mouse model showed that high levels of phospholipids are present in the serum [23–25]. In vitro, 31P NMR spectroscopy-based assessment of phospholipid metabolism is gaining importance among investigators of tumor pathophysiology and systemic cancer effects [26–31]. 31P NMR spectroscopy-based analysis of serum phospholipids in leukemia, lymphoma, renal cell carcinoma and other non-hematological cancers are of immense clinical importance [26–28]. These studies were the basis for NMR spectroscopy-based analysis of the phospholipids in the whole blood of DMD patients.

The present study was performed to evaluate quantitative changes in phospholipids (PL), i.e. phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and lyso-phosphatidylcholine (LyPC) in whole blood of DMD patients as compared to normal individuals.

Materials and methods

Blood samples (n = 32) of suspected DMD cases (Age, Mean ± SD; 8.0 ± 1.6 year; sex, male) were collected from outpatient clinics and inpatients wards at department of neurology Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India. Approval from Institute’s ethical committee was obtained before beginning of this study. Blood samples (n = 16) of healthy male volunteers (Age, Mean ± SD; 8.6 ± 2.3 year) were used as control. These samples were collected in EDTA (Ethylene diamine tetra acetate) coated vials and stored at −80 °C. The collected blood sample from each individual was two milliliters. Experimental analysis of blood samples was performed within a week from the time of collection.

Chemicals

All reagents were procured from the Sigma-Aldrich, USA.

Methods

Clinical examination

Diagnosis of suspected DMD patients was done at Neurology OPD (out-patients department), Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow. Patients were examined on the basis of clinical symptoms, signs and family history. Patients complained about their muscular wasting and weakness, difficulty in running or getting up from the ground and frequent falls or toe-walking. They also told that the beginning of symptoms took place in between the age of 3–5 years. Patients were examined for the well-known Gower’s sign (weakness of knee and hip extensors results in the classical Gower’s sign when the child attempts to rise from the floor) and valley sign along with calf muscle hypertrophy [1, 32]. Valley sign was helpful to diagnose chair bound patients as well as those patients, who did not have prominent calves [33] (Fig. 1a, b).

Fig. 1
figure 1

Clinical signs in DMD patients a Gower’s maneuver or sign, b valley sign—a valley (yellow) between the two mounts (red)

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Measurement of CK (creatine kinase) activity

The measurement of plasma CK activity is important for the diagnosis of muscle diseases such as muscular dystrophy, polymyositis, and muscle trauma [34, 36]. In present study, plasma CK activity was measured by the method of Rosalki et al. [36].

Gene mutation analysis

DNA was extracted by salting out method from 1.0 ml blood using standard protocols [3, 4, 37]. The concentration of DNA samples was determined and integrity of DNA in the samples was confirmed by 1.0 % (w/v) agarose gel electrophoresis. Gene mutation (deletion) analysis was performed by multiplex PCR using 21 pairs of primers (Pm 3, 4, 6, 8, 12, 17, 19, 34, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 55 and 60) of the dystrophin gene [8, 9]. The 10 µl aliquot of amplified products was loaded on 2 % agarose gel and electrophoresis run was performed. After complete separation of bands in gel electrophoresis, the gel was stained and bands were visualized under UV transilluminator.

Immunohistochemical analysis

Muscle biopsy of suspected DMD patients, who showed the negative result for gene mutation analysis, was performed for further confirmation of the diagnosis. Immunohistochemical staining of the dystrophin protein was carried out on the muscle specimen using standard protocol [38].

31P NMR spectroscopy-based analysis of blood

Processing of blood samples

Frozen blood samples were processed before performing the experiments of 31P NMR spectroscopy. The required reagents for processing were Lysis buffer (Sucrose, Triton X-100, MgCl2 and Tris), proteinase K buffer (NaCl and EDTA) and SDS (Sodium dodecyl sulfate) (10 %; pH= 7.2). All these reagents and frozen blood were mixed together for 2–3 min with the help of cyclomixer and then the whole mixture was incubated at 37.5 °C for 30 min. Processed samples were lyophilized. Lyophilized sample were re-dissolved in 0.5 ml deuterated water and transferred to 5 mm NMR tube. 31P NMR spectroscopy experiments were performed on Bruker Avance 400 MHz spectrometer (Bruker Biospin, Zurich, Switzerland) at 25 °C temperature.

Quantification of phospholipids

Quantification of phospholipids is based on the fact that the NMR signal intensity is proportional to the total amount of the chemical constituents present in the sample. NMR peak intensity can be calculated relative to the intensity of reference peak with the known concentration. Errors associated with the quantification are minimized by performing the experiment for all the samples at uniform experimental conditions [39, 40].

NMR spectroscopy-based precise and accurate quantification also depends upon two experimental factors : (1) “quantitative experimental conditions”, including selection of appropriate parameter such as relaxation delay and pulse sequence and (2) selection of suitable post acquisition processing parameters for the optimization of spectral integration [41].

31P NMR spectroscopy parameters

One-dimensional 31P NMR experiment was performed on processed blood using single pulse sequence. The parameters used were:—spectral width: 10,000 Hz; time domain size: 16 K; number of scans: 1000; relaxation delay: 10 s; line broadening: 10.00 Hz. These parameters were optimized and used to achieve the standard conditions for accurate quantification.

T1 (spin–lattice relaxation) measurement experiment for the relaxation delay of different phospholipids

Phosphorus T1 measurement experiment were performed on the whole processed blood (in deuterated water) with MDP (methylene di phosphonic acid) capillary with standard inversion recovery pulse sequence. The T1 value for PC, PE, PI, PS and LysPC were 1.32, 4.47, 5.4, 5.8 and 1.88 s, respectively. Relaxation time was measured by “Inversion Recovery Method” [42]. Quantification of the phospholipids can be measured by taking into the effect of incomplete relaxation during re-cycle delay. When the relaxation time is known, this can be used to predict the optimum pulse sequence cycle time for accurate quantification.

The amount of a phospholipid is calculated from the ratio of its peak area to that of the internal standard by the equation:

$$\frac{{M_{\text{t}}^{\text{P}} }}{{M_{\text{t}}^{\text{S}} }} = \frac{{M_{\infty }^{\text{P}} \left[ {1 - \exp \left( { - {\tau \mathord{\left/ {\vphantom {\tau {T_{1}^{\text{P}} }}} \right. \kern-0pt} {T_{1}^{\text{P}} }}} \right)} \right]}}{{M_{\infty }^{\text{S}} \left[ {1 - \exp \left( { - {\tau \mathord{\left/ {\vphantom {\tau {T_{1}^{\text{S}} }}} \right. \kern-0pt} {T_{1}^{\text{S}} }}} \right)} \right]}}$$

where superscripts P and S refer to the phospholipid and internal standard, respectively. The ratio of fully relaxed magnetization \({{M_{\infty }^{\text{P}} } \mathord{\left/ {\vphantom {{M_{\infty }^{\text{P}} } {M_{\infty }^{\text{S}} }}} \right. \kern-0pt} {M_{\infty }^{\text{S}} }}\) represents the true relative molar quantities of phosphorous in the lipid and the standard, and t is the repetition time [43].

Assignment of the phospholipids

Assignment of the phospholipids (PC, PE, PI, PS and LysPC) was confirmed by one-dimensional 31P NMR spectroscopy, using single pulse sequence, was performed in non-polar solvents (chloroform alone and with combination of chloroform and methanol) as well as on standard phospholipids in aqueous media [D2O with combination of Lysis buffer, proteinase K buffer and SDS (pH = 7.2)]. Assignments were also confirmed by literature [43, 44].

Quantitative evaluation of the phospholipids in blood samples (in duplicate)

Twelve blood samples of healthy individuals of different age groups were divided into two equal parts (0.5 ml by volume) and processed through above described method. One-dimensional 31P NMR spectroscopy-based experiment was performed on both parts of the processed blood samples and quantity of phospholipids (PC, PE, PI, PS and LysPC) was calculated. Both parts of the samples were close together on the basis of the quantity of PC, PE, PI, PS and LysPC. Uniform conditions were applied in sample processing and performing the NMR experiment to eliminate the possible errors.

Statistical analysis

Quantitative data of PC, PE, PI, PS and LysPC was not normally distributed. In this regard, median of PC, PE, PI, PS and LysPC in DMD and healthy subjects was compared by Mann–Whitney test for independent groups. The p value less than 0.05 was considered significant. The data management and analysis were carried out using statistical software SPSS version 15.0.

Results

Clinical examination

All suspected cases of DMD (n = 32) were clinically considered in the frame of DMD on the basis of clinical examinations, including symptoms, signs and family history. After preliminary examinations, confirmatory tests of DMD were done.

Measurement of CK (creatine kinase) activity

Activity of CK was measured for sixteen healthy subjects with the median value of 123.5 IU/L and situated in the range of 20–200 IU/L. Median value of CK activity was found 8529 IU/L with the range of 1183–29000 IU/L for clinically recognized thirty-two DMD patients.

Gene mutation analysis

Gene mutation analysis showed positive gene deletion in twenty-two cases of clinically recognized DMD patients. Deletion in exons 45–52 was observed in eighteen cases and four cases demonstrated only single deletion in exons 50 and 52. Negative results were observed in ten cases of clinically recognized DMD patients (Fig. 2).

Fig. 2
figure 2

Electrophoretic pattern in Lane-1, showed the deletion in exons 48 and 51, Lane-2 showed no deletion in exons and Lane-3 showed the deletion in exons 3 and 52 (C control, D disease)

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Immunohistochemical analysis

Immunohistochemical analysis of clinically recognized ten cases of DMD patients (showed negative result for gene mutation analysis) was performed on muscle biopsy specimens for dystrophin protein. Dystrophin protein was absent in the muscle of DMD patients (Fig. 3).

Fig. 3
figure 3

Dystrophin staining showed a presence of dystrophin in normal muscle, b completely absent of dystrophin in DMD muscle

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31P NMR spectroscopy-based analysis of blood

One-dimensional 31P NMR spectra of processed blood of DMD patients and normal subjects were obtained (Fig. 4). Quantitative analysis of these spectra showed that the phospholipids [PC, PE, PI, PS and LysPC] in the blood of DMD patients were significantly higher (p < 0.05) as compared to healthy/normal subjects (Table 1). There were no significant differences observed for the phospholipids in the blood of negative gene deletion cases of DMD patients as compared to DMD cases with positive gene deletion (p > 0.05) (Table 2). Quantity of phospholipids in negative gene deletion cases of DMD patients as well as DMD cases with positive gene deletion was significantly higher (p < 0.05) as compared to normal individuals (Table 3).

Fig. 4
figure 4

31P NMR spectra of the whole blood of DMD patients and healthy subjects showed the phospholipid components (PC phosphatidylcholine, PE phosphatidylethanolamine, PI phosphatidylinositol, PS phosphatidylserine, Lys-PC Lyso-phosphatidylcholine)

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Table 1 Evaluation of the quantity of phospholipids classes in DMD patients vs. healthy subjects
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Table 2 Evaluation of the quantity of phospholipids classes in gene deletion positive cases of DMD patients vs. negative gene deletion cases of DMD patients
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Table 3 Evaluation of the quantity of phospholipids classes in gene deletion positive cases of DMD patients, negative gene deletion cases of DMD patients as compared to normal subjects
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Discussion

Preliminary diagnosis of DMD patients were based on symptoms, family history and clinical signs (Gower’s sign and valley sign). Creatine Kinase estimation, gene mutation analysis and immunohistochemical analysis were done as confirmatory tests.

Quantitative alteration of phospholipids in blood samples of confirmed DMD patients were done using 31P NMR spectroscopy. Quantity of PC, PE, PI, PS and LysPC in blood of DMD patients was significantly higher as compared to normal subjects. The present finding is supported by various previous reports. The abnormal erythrocyte membrane in DMD patients has been reported earlier [21, 45–49]. There were biochemical, morphological and biophysical basis of alteration observed in red blood cell (RBC) membrane of DMD patients [21]. Structural abnormality of erythrocyte membranes is a result of its tendency to form abnormally oriented vesicles [45]. Abnormality in the structure of erythrocyte membrane in these patients was also confirmed by freeze-fracture studies [46]. Erythrocyte from DMD patients is fragile and interactions between skeletal proteins and/or between membrane and skeleton components may contribute to the alterations of erythrocyte membranes in DMD [47]. This is another evidence of an organizational abnormality in DMD erythrocyte membranes. Decreased water permeability of erythrocyte membranes in patients with DMD also provides the evidence of its abnormality [48]. DMD erythrocyte plasma membrane abnormality is also proved by the alteration in ion transport and in various enzymatic activities [49].

Alteration of phospholipids in blood of DMD patients have been reported earlier. Elevated level of plasma phospholipid was found in the serum of animals undergoing dystrophies in their muscles [24, 25]. Serum phospholipid was also found prominent in patients with DMD [50]. Hunter et al. [46] reported increased amount of PC in outer leaflet of erythrocytes membrane of DMD as compared to normal/control subjects. A study by Piperi et al. [51] showed abnormal fatty acid composition and disorganization of erythrocyte membrane in patients with DMD.

Collective findings of these two groups confirm that structural abnormality of erythrocyte membrane is related to the alteration of phospholipid composition. The major constituent of lipid bilayer of membrane is phospholipids [52] that are altered in blood plasma and plasma membrane of red blood cell. There are very few reports on alteration of different classes of phospholipid in blood of DMD patients. The present study attempts to cover the lacunae.

There are several other remarkable reasons responsible for the higher level of phospholipid classes in blood of DMD patients as described in the present results. During degeneration of the muscle membrane, phospholipids are released in the blood pool where they get accumulated. Muscle regeneration enhances synthesis of phospholipid [52]. Increased rate of synthesis of phospholipids and breakdown of muscle membrane may be responsible for increase level of phospholipids in blood of DMD patients [52, 53]. An earlier study demonstrated that increased rate of phospholipid metabolism in gastrocnemius muscle and sciatic nerves of dystrophic mouse model is due to elevated activity of phospholipases A [54]. Elevated activity of phospholipases A in muscles is also responsible for enhanced amount of phospholipids in the blood because higher activity of phospholipases A is also reported in human erythrocytes from patients with DMD [55].

There was no significant difference in different phospholipid classes in whole blood of negative gene deletion cases of DMD patients (diagnosis was confirmed by the method of immunohistochemistry on muscle tissue) as compared to DMD cases with positive gene deletion. The phospholipid classes in whole blood were elevated in negative gene deletion cases of DMD patients and DMD cases with positive gene deletion as compared with normal individuals. Elevated levels of different phospholipids in whole blood of negative gene deletion cases of DMD were similar to the positive gene deletion cases of DMD. The results of this study conclusively prove that 31P NMR spectroscopy-based quantitative comparison of different classes of phospholipids is more reliable and sensitive method as compared to gene mutation analysis for diagnosing DMD.

Conclusion

The present study distinguishes the patients with DMD from the healthy subjects on the basis of quantity of phospholipids in blood. These observations may be useful in future for the development of new diagnostic method of DMD.