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Metabolomic analysis of serum by (1) H NMR spectroscopy in amyotrophic
lateral sclerosis
Alok Kumara,
1,
Lakshmi Balab,
1,
Jayantee Kalitaa,
U.K. Misraa,
R.L. Singhc,
C.L. Khetrapalb
and G. Nagesh Babua,
, 
Received 9 November 2009;
revised 11 January
2010;
accepted 11 January 2010.
Available online 22
January 2010.
Abstract
Background
Amyotrophic lateral sclerosis (ALS), an invariably fatal neurological disorder shows complicated pathogenesis that poses challenges with respect to diagnosis as well as monitoring of disease progression.Methods
We investigated metabolite profiles in the serum of 30 patients with ALS, 10 patients of Hirayama disease, which served as a neurological disease control and 25 healthy controls by using (1) H NMR spectroscopy.Results
Compared to healthy controls, the ALS patients had higher quantities of glutamate (P < 0.001), beta-hydroxybutyrate (P < 0.001), acetate (P < 0.01), acetone (P < 0.05), and formate (P < 0.001), and lower concentrations of glutamine (P < 0.02), histidine (P < 0.001) and N-acetyl derivatives. On the other hand, Hirayama disease patients had significantly higher median concentrations of pyruvate (P < 0.05), glutamate (P < 0.001), formate (P < 0.05) and lower median concentrations of N-acetyl derivatives. Furthermore, we also found that serum glutamate showed a positive correlation (P < 0.001, r = 0.6487) whereas, histidine showed a negative correlation (P < 0.001, r = − 0.5641) with the duration of the disease in ALS.Conclusions
Such (1) H NMR study of serum may reveal abnormal metabolite patterns, which could have the potential to serve as surrogate markers for monitoring ALS disease progression.
Keywords: Motor neuron disease; N-acetyl
derivatives; Histidine; Ketones; Serum metabolites; Biomarkers
Abbreviations: ALS, Amyotrophic lateral sclerosis;
HD, Hirayama disease; TSP, 3-trimethylsilyl-(2,2,3,3-d4)-propionic
acid; DQF-COSY, Double quantum filtered correlation spectroscopy;
TOCSY, Total correlation spectroscopy; ROS, Reactive oxygen species;
BHBT, D-ß-hydroxybutyrate
Article Outline
1. Introduction
Amyotrophic lateral sclerosis (ALS; also known as motor neuron disease) consists of progressive, although variable degeneration of the corticospinal tract, brainstem, and spinal anterior horn neurons, with a markedly heterogeneous clinical presentation and course. One manifestation of this disease, despite a uniformly fatal outcome, is a wide range of survival times from a few months to several decades, with a consistent median of 2–4 y from onset of symptoms in population-based studies [1]. The idea that ALS is a typical example of a disease in which certain populations of neurons are “selectively vulnerable” to degeneration is becoming less tenable [2]. ALS is a syndrome, probably another “multiple system” neurodegenerative disorder, that has clinical and pathological overlap with fronto-temporal dementia that is yet to be fully defined [3]. A comprehensive model for the degeneration of motor neurons in ALS is needed, but contemporary hypotheses include aberrant axonal transport [4], protein aggregation, glutamate excitotoxicity and oxidative stress, apoptosis, mitochondrial dysfunction, and microglial activation [5]. Although linkage studies in a subset of familial cases have identified mutations in SOD1 (the gene that encodes copper–zinc superoxide dismutase-1), genetic association and genome-wide association studies in patients with sporadic ALS have not established a simple genetic model for ALS [6] and the disorder is best understood as a predominantly sporadic disease [7].Hirayama disease, used as a neurological disease control, is characterized by forward migration of the posterior surface of the dura mater with compression of the spinal cord on CT myelograms and/or MR images obtained with flexion of the cervical spine [8]. It is also known as juvenile muscular atrophy of the distal upper extremity, monomelic amyotrophy, benign focal amyotrophy [9], juvenile muscular atrophy of a unilateral upper extremity [10], and juvenile asymmetric segmental spinal muscular atrophy [11]. Muscular weakness and wasting of the distal upper extremity, predominantly in young men, is followed by spontaneous arrest within several years. Recently, in our study of Hirayama disease, survival motor neuron gene deletion was not found [12]. Dural sac displacement during neck flexion and the resulting cord compression are assumed to contribute to anterior horn cell damage.
2. Materials and methods
2.1. ALS patients and control subjects
ALS patients were selected using the El Escorial revised clinical criteria [13]. Evaluations included a detailed history and physical examination and extensive hematological, biochemical, electrophysiological and magnetic resonance imaging testing. The diagnosis of ALS required the presence of both upper and lower motor neuron signs, clear evidence of progression, normal nerve conduction velocities, late responses and electromyography evidence of diffuse denervation. Patients diagnosed with ALS also met an extensive list of exclusionary criteria, including sensory findings, unexplained bowel or bladder changes and toxic disorder that could mimic ALS, e.g. myelopathy, endocrine abnormalities or peripheral neuropathy.The diagnosis of Hirayama disease was carried out according to the criteria of Hirayama [14]. Thirty ALS and ten Hirayama disease patients were diagnosed and patient characteristics of ALS and Hirayama disease were given in Table 1. Only the definite ALS patients were included and all probable and laboratory supported probable patients were excluded from the present study. There were twenty five males and five females in ALS group and all males in Hirayama disease group. None of the selected patients were taking any medication including riluzole.
Table 1. Patient characteristics in ALS and
Hirayama disease.
| S.N | Parameter | Control (n = 25) | ALS (n = 30) | Hirayama disease (n = 10) |
|---|---|---|---|---|
| 1 | Age, n ys ± SD | 40 ± 20 | 42 ± 17 | 36 ± 10 |
| 2 | Male (%) | 80 | 83 | 100 |
| 3 | Non-smokers (%) | 92 | 83 | 90 |
| 4 | Vegetarian (%) | 60 | 70 | 70 |
| 5 | Speech abnormality (%) | 0 | 90 | 30 |
| 6 | Swallowing problem (%) | 0 | 70 | 0 |
| 7 | Neck injury (%) | 0 | 1 | 0 |
| 8 | Riluzole medication (%) | 0 | 0 | 0 |
| 9 | Atrophy in hand/leg (%) | 0 | 90 | 90 |
| 10 | Duration of illness in months ± SD | 0 | 12.40 ± 7.5 | 17.40 ± 7.7 |
Analysis of serum was performed in a controlled manner so that patient and control characteristics, e.g., age, gender, smoking and also their food habits shall not influence the metabolite analysis. In the questionnaires, the information regarding their diet was based on at least one week retrospective recall, because, certain diets can influence the concentrations of individual amino acids after a prolonged intake. Twenty five age- and sex-matched healthy volunteers without any neurological problems were used as controls and they were also free from any medication.
2.2. Preparation of samples
The laboratory personnel were maintained masked to the clinical diagnosis and group of the subjects, matching each blood sample by letter coding, and so were the clinicians to subsequent concentrations until the end of the study. This study was approved by the Institute's human ethics committee. After informed consent was obtained from all patients and control subjects, antecubital whole-blood samples were drawn from a peripheral vein using a 25-gauge needle in the morning hours (08:30–10:30) after an overnight fast and 15 min of supine rest. Blood collected in a serum separator tube was kept for 30 min. Serum from blood after clotting, was separated out and collected in a clean tube and again centrifuged for 10 min at 3000 rpm and frozen at − 80 °C until NMR analysis was performed. We have not obtained cerebrospinal fluid from patients and controls due to ethical considerations.2.3. Metabolic profile of serum by (1) H NMR
The NMR experiments were performed on Bruker Avance DRX 400 spectrometer equipped with a broad band inverse probehead with a Z-shielded gradient (Bruker Biospin, Karlsruhe, Germany). Deuterium oxide (D2O; 99.98 at.% D) and the sodium salt of 3-trimethylsilyl-(2,2,3,3-d4)-propionic acid (TSP) were from Sigma-Aldrich (Milwaukee, WI). For the quantitative estimation of various metabolites, 500 µl of the serum specimen from each subject was taken in a 5 mm NMR tube with a reusable sealed capillary tube containing 30 µL of 0.375% sodium salt of TSP in deuterium oxide before recording the spectra. One-dimensional (1)H NMR measurements were obtained using Carr–Purcell–Meiboom–Gill (CPMG) sequence [15] with water suppression by pre-saturation at 298 K. The parameters used were: spectral sweep width, 8000 Hz; data points, 32 K; flip angle of the radio frequency pulse was 90°; total relaxation delay of 15 s; T2 filtering was obtained with an echo time of 640 µs repeated 420 times resulting into total duration of effective echo time as 269 ms; scans, 128; window function, exponential; line broadening function, 0.3 Hz. The presence of comparatively broad peaks between 2.01 and 2.2 ppm in all the specimens drew our attention. The three equivalent N-acetyl protons of N-acetyl containing metabolites (chiefly N-acetyl aspartate, N-acetylneuraminic acid and N-acetyl glucosamine) have earlier been reported to resonate as a singlet in a small part of the (1) H NMR spectrum [16] and [17]. However, in addition a few metabolites without an N-acetyl group are also known to resonate in this region. These include glutamine (β-CH2 multiplet at 2.12 ppm), proline (β-CH2 multiplet at 2.02 ppm) and methionine (S-CH3 singlet at 2.13 ppm) [18]. In order to evaluate contributions from N-acetyl containing metabolites separate (1) H NMR spectra using Hahn spin–echo pulse sequence (90° x–τ–180° y–τ–collect) with a τ value of 68 ms were recorded for such purpose in all the serum specimens. This was performed to observe the phase reversal of J-coupled multiplets. The contributions of these resonances in each case were compared. Unambiguous assignments of various other metabolites were performed using two-dimensional double quantum filtered correlation spectroscopy (DQF-COSY) [19] and total correlation spectroscopy (TOCSY) [20] and spiking experiments using standard chemicals. For phase sensitive DQF-COSY, the following parameters were used: i) A total of 2048 data points were collected in the t2 domain over a spectral width of 8000 Hz. ii) The water resonance was presaturated during the total relaxation delay of 2.64 s. 512 time domain points were collected in t1 with 64 acquisitions. For TOCSY the parameters used were: data points — 2 k, spectral width — 8000 Hz, total relaxation delay — 1.63 s, and spin lock time — 70 ms. 512 time domain data points were collected in t1 using 88 acquisitions. The resulting 2D data were Fourier transformed after zero filling in t1-dimension to 1024 points and multiplying in both the dimensions by squared sine bell window function shifted by 90°. The procedure followed for quantification of the metabolites was the same as reported earlier [21].2.4. Statistical analysis
Data were expressed as mean ± SD. To improve the precision of the data and find out the influence of confounding factors (age, sex, smoking and disease duration) on the metabolite concentrations, we created sub-groups and analyzed the data by stratified sampling. Univariate statistical differences between ALS, Hirayama and control serum metabolites were analyzed using the Mann–Whitney U test. Mean significant differences in more than two groups were analyzed using one way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. A P < 0.05 was considered to be statistically significant. Correlations between the duration of illness and the concentrations of metabolites were calculated using a nonparametric two-tailed Spearman's correlation test. These analyses were done using the SPSS statistical software, version 12.0 (SPSS Inc., Chicago, IL).3. Results
The data reported in patients as well as normal controls were the result of duplicated measurements of NMR in the individual patients. Following stratified sampling analysis, we did not observe any variation in the metabolite concentrations between the sub-groups with respect to confounding factors; age (> 40 y vs. < 40 y, sex (males vs. females) or smoking (smokers vs. non-smokers)) among ALS/Hirayama disease or controls. Hence, these data were not tabulated sub-group wise.As demonstrated by (1) H NMR spectroscopy, serum glutamate (P < 0.001), β-hydroxybutyrate (BHBT) (P < 0.001), acetate (P < 0.01), acetone (P < 0.05), and formate (P < 0.001) concentrations were significantly higher in the patients of ALS with respect to age and sex-matched control subjects. On the other hand, serum concentrations of glutamine (P < 0.02) and histidine (P < 0.001) were significantly decreased in ALS with respect to healthy controls. Whereas, the serum concentrations of alanine, lysine, pyruvate, citrate, glucose, creatine/creatinine and tyrosine in ALS patients were comparable to control concentrations.
In Hirayama disease patients, it was found that glutamate (P < 0.01), pyruvate (P < 0.05) and formate (P < 0.05) were increased significantly in comparison to control subjects, whereas, BHBT, acetate, acetone, formate, alanine, lysine, citrate, glucose, creatine/creatinine and tyrosine concentrations showed no significant change (Table 2).
Table 2. Serum metabolite profiles in ALS and
Hirayama disease patients as revealed by (1) H NMR spectroscopy.
Data
are expressed in median and (range) ± standard deviation. •••P < 0.001,
••P < 0.01, •P < 0.05
ALS vs. control; ***P < 0.001, *P < 0.05
ALS vs. Hirayama disease; ■■P < 0.01, ■P < 0.05
Hirayama disease vs. control.
| S.N | Metabolites (μmol/l) | Control (n = 25) | ALS (n = 30) | Hirayama disease (n = 10) |
|---|---|---|---|---|
| 1 | BCAA | 183 (21–1698) ± 309 | 194 (24–1527) ± 296 | 210 (132–260) ± 49 |
| 2 | BHBT | 0 | 2 (0–78) ± 17••• *** | 0 |
| 3 | Lactate | 1345 (168–3645) ± 740 | 1368 (575–7261) ± 1370 | 1364 (493–2743) ± 743 |
| 4 | Alanine | 408 (31–733) ± 132 | 433 (45–1715) ± 282 | 458 (315–640) ± 114 |
| 5 | Lysine | 125 (62–155) ± 25 | 96 (0–398) ± 75 | 86 (0–391) ± 129 |
| 6 | Acetate | 8 (5–18) ± 4 | 15 (0–75) ± 20•• | 16 (0–48) ± 17 |
| 7 | Acetone | 9 (0–23) ± 6 | 15 (4–160) ± 41•* | 9 (0–27) ± 10 |
| 8 | Pyruvate | 0 (0–94) ± 20 | 0 (0–1100) ± 256 | 41 (0–165) ± 68■ |
| 9 | Glutamine | 448 (359–665) ± 88 | 406 (273–3791) ± 754 | 401 (260–528) ± 112• |
| 10 | Glutamate | 0 (0–225) ± 68 | 92 (0–1861) ± 386••• | 112 (0–234) ± 74■■ |
| 11 | Citrate | 46 (0–96) ± 25 | 35 (0–271) ± 54 | 45 (0–264) ± 91 |
| 12 | Creatine/creatinine | 30 (0–61) ± 14 | 43 (0–115) ± 27 | 34(11–295) ± 102 |
| 13 | Glucose | 3723 (948–32410) ± 5797 | 4492 (0–10346) ± 2227 | 3887 (3280–4902) ± 524 |
| 14 | Histidine | 67 (48–85) ± 10 | 55 (17–210) ± 38••• | 61 (28–72) ± 18 |
| 15 | Tyrosine | 0 (0–27) ± 10 | 3 (0–122) ± 25 | 12(0–32) ± 12 |
| 16 | Formate | 0 (0–16) ± 6 | 16 (0–69) ± 15••• | 11 (0–34) ± 11■ |
However, the concentration indices of two metabolites which were distinctly altered in ALS patients compared to healthy controls were further found to have a significant correlation with the duration of the disease but not with age, sex or other characteristics. Of these, glutamate showed a positive correlation (P < 0.001, r = 0.6487; Fig. 1) whereas, histidine showed a negative correlation (P < 0.001, r = −0.5641; Fig. 2) during the course of ALS. Representative (1) H NMR spectra of serum were depicted from control subjects (Fig. 3a), patients with Hirayama disease (Fig. 3b), and patients with ALS (Fig. 3c).
 | Full-size image (9K) |
Fig. 1. Glutamate concentration (µmol/l) in the serum of ALS patients vs. duration of illness (total of 30 ALS patients were represented at 6, 12 and 24 months following the onset of illness). Controls (n = 25) are represented at time zero. Each marker represents one individual.
 | Full-size image (9K) |
Fig. 2. Histidine concentration (µmol/l) in the serum of ALS patients vs. duration of illness (total of 30 ALS patients were represented at 6, 12 and 24 months following the onset of illness). Controls (n = 25) are represented at time zero. Each marker represents one individual.
 | Full-size image (39K) |
Fig. 3. Representative serum NMR spectra of control subjects (a), Hirayama disease patients (b) and ALS patients (c).
4. Discussion
Glutamate is a powerful amino acid neurotransmitter that plays a pivotal role in the formation of synapses and neuronal circuitry, long-term potentiation and depression, and both normal learning and addictive behavior. On the other hand, it was found that specific type of excitotoxicity triggered by the amino acid glutamate which is a key mechanism implicated in the mediation of neuronal death of ALS [22], [23] and [24]. In the present study, we noticed a significant elevation of the metabolite glutamate in both ALS and Hirayama disease as revealed by (1) H NMR spectroscopy. These higher glutamate signals are consistent with the hypothesis of glutamate excitotoxicity in ALS pathogenesis. On the other hand, glutamine concentrations decreased in ALS only, which might represent the imbalance between glutamate–glutamine conversion cycle that occurs in post synaptic buttons and astrocytes during excitotoxicity. Earlier study of Pioro [25] also showed in vivo evidence of abnormal glutamate metabolism in the CNS parenchyma of patients with ALS.One more metabolite of interest, formate, increased in both ALS and Hirayama disease. Formate is produced as a byproduct in the production of acetate. It is responsible for both metabolic acidosis and disrupting mitochondrial electron transport and energy production by inhibiting cytochrome oxidase activity, the terminal electron acceptor of the electron transport chain [26]. Cell death from cytochrome oxidase inhibition by formate is believed to result partly from depletion of ATP, reducing energy concentrations so that essential cell functions cannot be maintained. Furthermore, inhibition of cytochrome oxidase by formate may also cause cell death by increased production of cytotoxic reactive oxygen species (ROS) secondary to the blockade of the electron transport chain.
Another metabolite histidine which is considered to be an antioxidant, significantly decreased in our study. An imidazole ring in the histidine molecule helps in scavenging ROS generated by cells during the acute inflammatory response [27]. This agent also protected cultured rat primary neurons against oxidative and hypoxic injury [28]. It is also reported that thioperamide with l-histidine preserved SOD activity in the ischemic hemisphere [29]. This seems to be caused by the preservation of the endogenous scavenging system due to l-histidine that neutralizes oxygen-free radicals. It is also reported that low plasma concentrations of histidine are associated with protein-energy wasting, inflammation, oxidative stress and greater mortality in chronic kidney disease patients [30].
We also observed that ketone metabolites, d-ß-hydroxybutyrate (BHBT) and acetone were increased in the patients of ALS, but not in Hirayama disease. Earlier studies showed that BHBT protected neurons in the models of Alzheimer's and Parkinson's disease [31]. Ketone bodies not only reduce mitochondrial [NAD]/[NADH] but increase mitochondrial [Q]/[QH2] [32] and [33]. In the patients of ALS, continuous energy is required for the muscle and brain, so that carbohydrate and fats are continuously utilized as a source for energy, which may be responsible for significantly higher amounts of ketone bodies (BHBT and acetone) in the serum of patients with ALS. However, in the present group of patients we observed increased concentration of oxidative stress parameters and very low concentration of glutathione in the erythrocytes as the disease progressed [34] which implies that the elevated BHBT and acetone could not offer significant protection in the ALS patients.
Further, we also found that two other important metabolites pyruvate and acetate were increased in ALS patients and Hirayama disease. The fate of the major product of glycolysis, the cytosolic pyruvate, depends on several factors; some of which possibly being related to the cellular energy charge and others to the action of metabolite shuttles to maintain neurotransmitters, ammonia and carbon homeostasis among domains in the cell and its surroundings. In the same manner acetate is also increased to meet the required amount of ATP in the cell.
In the present study, N-acetyl derivative (N-acetyl-X) concentrations such as N-acetylaspartate (NAA) etc. decreased (but could not be quantified in definite terms). NAA reductions are related to energy impairment [35] and [36]. Though it was reported that the NAA content of the motor cortex, brainstem and corticospinal tract of ALS patients was reduced [37] and [38], compared with controls and the degree of reduction of NAA was related to the severity of upper motor neuron abnormalities [38], it could not be conclusively demonstrated here because of methodological limitations.
We can only speculate at this stage that the metabolic imbalances observed in the periphery may represent similar changes in the CNS. Though we did not use CSF in our study due to ethical considerations, there were previous studies by authors, who measured glutamate by conventional biochemical means and found that there were decreased glutamate concentrations in CNS tissue and increased concentrations in the serum and CSF of ALS patients, hence, proposed a hypothesis suggesting an imbalance in the intracellular vs. extracellular glutamatergic neurotransmitter system [39] and [40]. Glutamate excitotoxicity exacerbates the formation of ROS, which may be responsible for the oxidant–antioxidant imbalance, such as increased BHBT and acetone and decreased concentrations of histidine. Formate was also significantly increased due to ROS, which leads to the disruption in mitochondrial electron transport and energy production. Pyruvate and acetate were also increased, apparently to meet the requirement of ATP. On the other hand, N-acetyl-X derivatives decreased, which results in energy impairment.
Thus, by employing a sophisticated technique, viz. (1) H NMR spectroscopy, we were able to observe changes in some serum metabolite concentrations of ALS and Hirayama disease. Though the differences were statistically significant for certain metabolites, there was some overlap between the groups, especially glutamate, which is a general neurotransmitter. Hence, it would be very difficult to prove its specificity for each disease. However, in this regard, it may be noted that ALS etiology has been shown to have a multifactorial and multisystem involvement; therefore, wide variations in the metabolite concentrations are inherent part of this kind of study and there is a definite need to further elaborate the sensitive and specific metabolic signatures by other analytical means.
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Corresponding author. Department of Neurology,
Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli
Road, Lucknow 226014, UP, India. Tel.: + 91 522 2668008x2166; fax:
+ 91 522 2668017.1 Alok Kumar and Lakshmi Bala equally contributed to the paper.
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