Diagnosis and treatment of fibromyalgia (FM) remains a challenge owing to the lack of reliable biomarkers. Our objective was to develop a rapid biomarker-based method for diagnosing FM by using vibrational spectroscopy to differentiate patients with FM from those with rheumatoid arthritis (RA), osteoarthritis (OA), or systemic lupus erythematosus (SLE) and to identify metabolites associated with these differences. Blood samples were collected from patients with a diagnosis of FM (n ‫؍‬ 50), RA (n ‫؍‬ 29), OA (n ‫؍‬ 19), or SLE (n ‫؍‬ 23). Bloodspot samples were prepared, and spectra collected with portable FT-IR and FT-Raman microspectroscopy and subjected to metabolomics analysis by ultra-HPLC (uHPLC), coupled to a photodiode array (PDA) and tandem MS/MS. Unique IR and Raman spectral signatures were identified by pattern recognition analysis and clustered all study participants into classes (FM, RA, and SLE) with no misclassifications (p < 0.05, and interclass distances > 2.5). Furthermore, the spectra correlated (r ‫؍‬ 0.95 and 0.83 for IR and Raman, respectively) with FM pain severity measured with fibromyalgia impact questionnaire revised version (FIQR) assessments. Protein backbones and pyridine-carboxylic acids dominated this discrimination and might serve as biomarkers for syndromes such as FM. uHPLC-PDA-MS/MS provided insights into metabolites significantly differing among the disease groups, not only in molecular m/z ؉ and m/z ؊ values but also in UV-visible chromatograms. We conclude that vibrational spectroscopy may provide a reliable diagnostic test for differentiating FM from other disorders and for establishing serologic biomarkers of FM-associated pain.

Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent Km values of ∼50 μm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent Vm values of 24.5 and 17.5 μmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (Ki ∼20 μm). The predominant band on a gel at ∼19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells.

Spinal nerve ligation results in dramatic changes in spinal cord primary C-afferent fibers, which include atrophy with an accompanied decrease in calcitonin-gene-related peptide (CGRP). These changes parallel the activation of astrocytes, which have been implicated in the ensuing neuropathic pain states. As part of an effort to elucidate the role of the downstream effectors of astrocyte reactivity in the context of allodynia, the expression of fibroblast growth factor-2 (FGF-2) was examined following tight ligation of L5 and L6 spinal nerves. FGF-2 is a pleiotropic cytokine that is synthesized and secreted by neurons and astrocytes. FGF-2 immunoreactivity was increased in ipsilateral dorsal horn reactive astrocytes at 1 and 3 weeks following nerve ligation. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) of laser-captured dorsal spinal cord sections revealed an increase in FGF-2 mRNA in the dorsal horn ipsilateral to nerve injury compared to contralateral and SHAM. Furthermore, an increase in FGF-2 mRNA in ispilateral dorsal root ganglia (DRG) was seen by in situ hybridization. These results demonstrate that, in response to ligation-induced injury of sensory neurons, FGF-2 is upregulated in both DRG neurons and in spinal cord astrocytes, suggesting neurotrophic functions of this growth factor following peripheral nerve lesion and possibly in astrocyte-related maintenance of pain states.

Fibroblast growth factor 1 (FGF-1, also known as acidic FGF) is a mitogen for a variety of mesoderm-and neuroectoderm-derived cells, as well as an angiogenic factor in vivo. It has been implicated in angiogenic diseases including atherosclerosis, cancer and inflammatory diseases. In the present study, the entire transcriptional unit of the mouse FGF-1 gene, including four promoters, is characterized. By nucleotide sequence and RNase protection analyses, we have determined that its 3-end resides 3.2 kilobase pairs downstream from the stop codon. We have previously cloned and characterized the mouse homologue of the human 1B promoter, as well as a novel upstream untranslated exon. In order to elucidate the regulatory mechanism of FGF-1 gene expression, the mouse promoter containing TATA and CAAT consensus sequences (FGF-1.A) was isolated from a P1 library and characterized. We further determined that the mouse heart is the most abundant source for the FGF-1.A mRNA. Finally, via both RNase protection analysis and 5-rapid amplification of cDNA ends, we determined the transcription start site of the FGF-1.A mRNA.

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