Establishment of a Measurement System for Sphingolipids in the Cerebrospinal Fluid Based on Liquid Chromatography-Tandem Mass Spectrometry, and Its Application in the Diagnosis of Carcinomatous Meningitis.

OBJECTIVES: Sphingolipids have been demonstrated to be involved in many human diseases. However, measurement of sphingolipids, especially of sphingosine 1-phosphate (S1P) and dihydro-sphingosine 1-phosphate (dhS1P), in blood samples requires strict sampling, since blood cells easily secrete these substances during sampling and storage, making it difficult to introduce measurement of sphingolipids in clinical laboratory medicine. On the other hand, cerebrospinal fluid (CSF) contains few blood cells. Therefore, we attempted to establish a system based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the measurement of sphingolipids in the CSF, and applied it for the diagnosis of carcinomatous meningitis. METHODS: We developed and validated a LC-MS/MS-based measurement system for S1P and dhS1P and for ceramides and sphingosines, used this system to measure the levels of these sphingolipids in the CSF collected from the subjects with cancerous meningitis, and compared the levels with those in normal routine CSF samples. RESULTS: Both the measurement systems for S1P/dhS1P and for ceramides/sphingosines provided precision with the coefficient of variation below 20% for sphingolipids in the CSF samples. We also confirmed that the levels of S1P, as well as ceramides/sphingosines, in the CSF samples did not increase after the sampling. In the CSF samples collected from patients with cancerous meningitis, we observed that the ratio of S1P to ceramides/sphingosine and that of dhS1P to dihydro-sphingosine were higher than those in control samples. CONCLUSIONS: We established and validated a measurement system for sphingolipids in the CSF. The system offers promise for being introduced into clinical laboratory testing.

Disturbance in cerebral spinal fluid sphingolipid content is associated with memory impairment in subjects infected with the human immunodeficiency virus.

Despite widespread use of antiretroviral therapies to control replication of the human immunodeficiency virus (HIV), dysfunctions of cognition that are collectively termed HIV-associated neurocognitive disorders (HAND) still occur in approximately 50% of those infected by the virus. Currently there is not a biomarker that can identify HIV-infected people who are at risk for the development of HAND. Previous studies have identified particular sphingolipid species that are dysregulated in HAND, but the neurocognitive correlates of these biochemical findings are not currently understood. To address this question, we compared cerebrospinal fluid (CSF) levels of sphingomyelin, ceramide, and sterol species with performance on standard neurological tests designed to assess the function of multiple cognitive and motor domains in HIV-infected subjects. We found that sphingomyelin:ceramide ratios for acyl chain lengths of C16:0, C18:0, C22:0, and C24:0 were associated with worse performance on several indices of memory. The most striking finding was for the acyl chain of C18:0 that consistently associated with performance on multiple tests of memory. These findings suggest that the sphingomyelin:ceramide ratio for C18:0 may be a reasonable surrogate marker for memory dysfunction in HIV-infected subjects.

Astroglial expression of ceramide in Alzheimer's disease brains: a role during neuronal apoptosis.

Accumulating evidences indicate that ceramide is closely involved in apoptotic cell death in neurodegenerative disorders and aging. We examined ceramide levels in the cerebrospinal fluid (CSF) or brain tissues from patients with neurodegenerative disorders and the mechanism of how intra- and extracellular ceramide was regulated during neuronal apoptosis. We screened the ceramide levels in the CSF of patients with neurodegenerative disorders, and found that ceramide was significantly increased in patients with Alzheimer's disease (AD) than in patients with age-matched amyotrophic lateral sclerosis (ALS) and other neurological controls. With immunohistochemistry in AD brains, ceramide was aberrantly expressed in astroglia in the frontal cortices, but not detected in ALS and control brains. To explore for the regulation of ceramide in astroglia in Alzheimer's disease brains, we examined the metabolism of ceramide during neuronal apoptosis. In retinoic acid (RA)-induced neuronal apoptosis, RA slightly increased de novo synthesis of ceramide, but interestingly, RA dramatically inhibited conversion of [14C] ceramide to glucosylceramide (GlcCer), suggesting that the increase of ceramide mass is mainly due to inhibition of the ceramide-metabolizing enzyme GlcCer synthase. In addition, a significant increase of the [14C] ceramide level in the culture medium was detected by chasing and turnover experiments without alteration of extracellular [14C] sphingomyelin levels. A 2.5-fold increase of ceramide mass in the supernatant was also detected after 48 h of treatment with RA. These results suggest a regulatory mechanism of intracellular ceramide through inhibition of GlcCer synthase and a possible role of ceramide as an extracellular/intercellular mediator for neuronal apoptosis. The increased ceramide level in the CSF from AD patients, which may be derived from astroglia, raises a possibility of neuronal apoptosis by the response to intercellular ceramide in AD.