Lysophosphatidic acid is associated with neuropathic pain intensity in humans: An exploratory study

The underlying mechanisms of neuropathic pain remain to be elucidated. Basic animal research has suggested that lysophosphatidic acids, which are bioactive lipids produced by autotaxin from lysophosphatidylcholine, may play key roles in the initiation and maintenance of neuropathic pain. Here, we investigated the clinical relevance of lysophosphatidic acids signaling on neuropathic pain in humans. Eighteen patients who had been diagnosed with neuropathic pain with varied etiologies participated in the study. Cerebrospinal fluid samples were obtained by lumbar puncture and the concentrations of 12 species of lysophosphatidic acids and lysophosphatidylcholine, autotaxin, and the phosphorylated neurofilament heavy subunit were measured. Pain symptoms were assessed using an 11-point numeric rating scale and the Neuropathic Pain Symptom Inventory regarding intensity and descriptive dimensions of neuropathic pain. The total lysophosphatidic acids were significantly associated with both pain intensity and symptoms. 18:1 and 20:4 lysophosphatidic acids in particular demonstrated the most correlations with dimensions of pain symptoms. Autotaxin and the phosphorylated neurofilament heavy subunit showed no association with pain symptoms. In conclusions, lysophosphatidic acids were significantly associated with pain symptoms in neuropathic pain patients. These results suggest that lysophosphatidic acids signaling might be a potential therapeutic target for neuropathic pain.


Introduction
The International Association for the Study of Pain (IASP) defines neuropathic pain as "pain caused by a lesion or disease of the somatosensory nervous system". Neuropathic pain is a debilitating condition that commonly impairs activities of daily living and health-related

Materials and methods
The study protocol was approved by the Institutional Review Board of the Ethics Committee of The University of Tokyo (approval number: 10516). We invited patients to participate in this study after they were referred to us by our outpatient clinic. All participants provided written informed consent. The three inclusion criteria were as follows: presence of neuropathic pain as diagnosed by the IASP [14]; aged at least 20 years old and able to provide informed consent; and no other neurological or psychological impairments. The details of the IASP neuropathic pain have been described elsewhere [14]. Briefly, patients were diagnosed with neuropathic pain when their pain had a neuroanatomically plausible distribution; there was a history suggestive of a relevant lesion or disease affecting the peripheral and/or central somatosensory nervous system; a distinct neuroanatomically plausible distribution was demonstrated by at least one neurological examination that confirmed negative or positive signs of somatosensory nervous system impairment; and presence of the relevant lesion or disease was demonstrated by at least one confirmatory test (e.g., imaging or electrophysiological studies). We considered the intensity of neuropathic pain as a study-inclusion criterion (i.e., numeric rating scale (NRS) value > 1 on an 11-point NRS). The duration of neuropathic pain was not included as an inclusion criterion, because the time course of LPA and LPC was not fully elucidated. Furthermore, we did not include the location of neuropathic pain, because it was unclear whether the location of a lesion affects LPA, LPC, or autotaxin concentrations in CSF.
The three exclusion criteria were as follows: inability to understand the questions in Japanese without any help; inability to provide informed consent; and altered level of consciousness. All participants provided CSF samples, which were collected by lumbar punctures of the L3/L4 or L4/L5 interspace. Participants were asked to quantify the average and maximum intensity of their neuropathic pain over the last week on an 11-point NRS, where "0" and "10" indicated "no pain" and "pain as bad as you can imagine", respectively. Neuropathic pain symptoms were assessed with the Japanese version of the NPSI [15,16], which assesses ten neuropathic symptoms corresponding to five different dimensions (i.e., symptom combinations) on an 11-point NRS. The NPSI dimensions are "burning (superficial) spontaneous pain", "pressing (deep) spontaneous pain", "paroxysmal pain", "evoked pain", and "paresthesia/dysesthesia"; severity of neuropathic pain can be evaluated based on the nature of pain. Mental status was assessed using the Hospital Anxiety and Depression Scale (HADS), which consists of seven questions regarding anxiety (HADS-A) and depression (HADS-D).

Laboratory analyses
The CSF samples were mixed and sonicated with a 10-fold volume of methanol and an internal standard. After centrifugation at 21,500 � g, the resulting supernatant was recovered and used for the LC-MS analysis. Then, 20 μl of methanol extract was separated using Nanospace LC (Shiseido) equipped with a C18 CALL PAK ACR column (1.5 � 250 mm; Shiseido) using a gradient of solvent A (5 mM ammonium formate in water) and solvent B (5 mM ammonium formate in 95% [v/v] acetonitrile). Elution was sequentially ionized using an ESI probe, and the parent ion (m/z 380.2) and the fragment ion (m/z 264.2) were monitored in the positive mode using the Quantum Ultra Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific). For LPA and LPC molecular species, 12 acyl chains (14:0, 16:0, 16:1, 18:0, 18:1, 18 :2, 18:3, 20:3, 20:4, 20:5, 22:5, and 22:6) were monitored respectively. In the present study, we extracted lipids under a neutral condition, and we did not separate 1-acyl-2-lyso-and 2-acyl-1-lyso-phospholipids. A more detailed description of measuring lysophospholipids is available in our previous article [17]. Briefly, we calculated the concentrations of lysophospholipids from the area ratio to the internal standard as follows: 1 μM 17:0 LPA for LPA species or 10 μM 17:0 LPC for LPC species. The autotaxin antigen levels in the CSF were determined using a two-site immunoenzymetric assay with an autotaxin assay reagent and the TOSOH AIA system (TOSOH, Tokyo, Japan) [18].
In addition to LPA, LPC, and autotaxin, we measured the phosphorylated neurofilament heavy subunit (pNF-H), a known biomarker of the severity of neuronal damage. pNF-H is a major structural protein in CNS axons, and increased pNF-H levels in CSF are observed in patients with the lumbar canal stenosis, which reflects severity of clinical symptoms (e.g., intermittent claudication and leg pain) [19]. CSF pNF-H levels were determined using a commercially available enzyme-linked immunosorbent assay kit (Human Phosphorylated Neurofilament H ELISA; BioVendor, Modrice, Czech Republic) according to the manufacturer's protocol. The CSF samples were diluted threefold before analysis. pNF-H positivity was defined by levels of >70.5 pg/ml [20].

Statistical analysis
All the data were statistically analyzed using R version 3.4.0 (The R Foundation for Statistical Computing, Vienna, Austria). The results are expressed as mean (standard deviation) or median (interquartile range) as appropriate. Correlations between measurements and clinical symptoms were tested using Spearman's correlation test, because the normality of many variables, especially the laboratory measurements in the CSF, was rejected by the Kolmogorov-Smirnov test. Because this was an exploratory study to elucidate the role of LPA and others on neuropathic pain in human subjects, we did not conduct any multivariate analyses. P-values less than 0.05 were regarded as statistically significant in all the analyses.
Four of the six correspondent species of LPA and LPC (16:0, 18:1, 18:2, and 20:4) showed a significant correlation (Fig 4). As for the LPC species, most of them (16:0, 16:1, 18:1, 18:2, and 20:4 LPC) exhibited a relationship with different pain measurements. However, the total LPC was not associated with any pain scores, except for the pressing pain scores. Autotaxin was entirely unrelated to any laboratory measurements and any pain symptoms. Furthermore, pNF-H showed no association with any LPA and LPC measurements or clinical parameters. Mental status (anxiety and depression) showed no correlation with the laboratory measurements except 18:0 LPA.

Discussion
Average pain intensity and total NPSI scores were linearly correlated with the total amount of LPA. Regarding the molecular species of LPA, 16:0, 16:1 and 18:1 LPA were correlated with average pain intensity, but the other LPA species were not. Different LPA molecular species showed correlations with different dimensions of the NPSI. Most of the correspondent species of LPA and LPC (16:0, 18:1, 18:2, and 20:4) were correlated with one another. Some LPC Our findings indicate that LPA is profoundly associated with neuropathic pain in human subjects, which follows previous findings from animal neuropathic pain models [9]. Focusing on the linear correlations between the CSF LPA and LPC species, LPA concentrations were relatively higher than LPC concentrations (Fig 4). This conflicts with findings that serum LPA concentrations were lower than serum LPC concentrations in patients with acute coronary syndrome [21]. Possibly because LPA is derived from LPC via the enzymatic activity of autotaxin and autotaxin is rich in the CSF [22], LPA might be produced efficiently in the CSF. Despite previous findings that nerve damage can increase LPC [12], the total amount of LPC and autotaxin were not associated with pain intensity in this study. Therefore, LPC and autotaxin by themselves may not have the potential to induce neuropathic pain, but may simply mediate the induction of neuropathic pain through LPA production [10].
We found that four of the measured LPA species, but not 18:0 and 18:2 LPA, were associated with the total NPSI score and the NPSI dimensions. Different LPA species activate LPA receptor subtypes with varied efficacies [11]. Among the LPA receptor subtypes, LPA1 and Table 2. Relationships of pain scores and laboratory measurements between female and male.

Female (n = 11) Male (n = 7) p value
Age (  auxiliary LPA3 are known to play a crucial role in demyelination and neuropathic pain [8]. 18:1 LPA demonstrated the most correlations with neuropathic pain measurements (average pain intensities, the total NPSI score, and 3 out of 5 NPSI dimensions). The 18:1 LPA molecular species has been revealed as key in animal neuropathic pain models, and is the ligand predominantly responsible for LPA1 and LPA3 receptor-mediated amplification of LPA production through microglial activation [13]. Other than 18:1 LPA, 16:0, 16:1 and 20:4 LPA demonstrated correlations with some dimensions of the NPSI. These LPA species have been reported to have a relatively high agonist potency on LPA1 and LPA3 receptors, secondary to 18:1 LPA, in a neuropathic pain animal model [13]. Supporting evidence has been derived Gray shadows indicate statistically significant correlations (p < 0.05).
https://doi.org/10.1371/journal.pone.0207310.t003  relatively low according to calcium mobilization assay [13], and possibly thereby 18:0 LPA and 18:2 LPA did not show any correlations with neuropathic pain in this study. The axonal damage biomarker, pNF-H, was not correlated with any LPA or LPC species in the present study. LPA1 receptor activation reportedly mediates the downregulation of myelin proteins, and nerve injury-induced downregulation of myelin proteins leads to the demyelination of the peripheral nerve [24,25]. In contrast, LPA activation could upregulate axonal growth followed by central sprouting of myelinated fibers in the spinal dorsal horn [26]. Such contradictory effects of LPA on myelin and axons might explain the present pNF-H results.
With respect to the nature of pain (i.e., different dimensions of the NPSI), patients with certain pain syndromes frequently select characteristic words to describe their pain. For example, cancer pain patients consistently characterize their pain as shooting, sharp, dull, and heavy [27], whereas patients with neuropathic pain tend to describe their pain as burning, shooting, tingling, and piercing [28]. We previously demonstrated that one of the two categories of neuropathic pain descriptions are more responsive to mirror visual feedback treatment than the other category [29]. Thus, the nature of pain is considered useful for understanding the underlying pathophysiological mechanisms of pain and forecasting analgesic responses to treatment [30]. In the present study, different LPA and LPC species were associated with different dimensions of the NPSI. Therefore, LPA and maybe LPC species might have respective pathophysiological mechanisms of neuropathic pain. Among the NPSI dimensions, only superficial spontaneous pain (i.e., burning pain) was weakly correlated with 18:1 LPA only. Burning pain is a major characteristic of neuropathic pain, and so future work should explore other LPA and LPC species or other explanatory mechanisms that could be linked to burning pain.
We investigated the influence of other factors such as gender and age on the LPA concentrations and pain symptoms. Table 2 demonstrated that male patients showed higher LPA concentrations than female. Male also had higher pain scores than female, which would affect the high levels of LPA concentrations in male patients. Regarding the factor of age, this study demonstrated age was not associated with either LPA concentrations or pain scores as shown in Table 3. Possible confounding factors other than gender and age (e.g. types and duration of diseases) might underlie the significant relationships of LPA concentrations and pain scores. However, this is an exploratory study and the sample size was relatively small. Therefore, further research is required to provide more detailed analysis and elucidate the exact mechanism of LPA and chronic pain.
In this study, we revealed for the first time that LPA concentrations are significantly correlated with clinical symptoms in patients with neuropathic pain. This supports findings from basic investigations that LPA signaling is the definitive mechanism of neuropathic pain. Autotaxin is reportedly involved in the development of neuropathic pain; however, we found no relationship between autotaxin and neuropathic pain symptoms.
Supporting information S1 Appendix. The dataset provided in this study.