Increased monohexosylceramide levels in the serum of established rheumatoid arthritis patients.

OBJECTIVES: To identify serum sphingolipids that could act as candidate biomarkers in RA. METHODS: We performed lipidomic analyses in the serum of 82 participants: 19 established RA patients, 18 untreated early RA patients, 13 untreated early arthritis patients not fulfilling the classification criteria for RA, 12 established SpA patients and 20 controls. We compared the lipid levels from the different patient groups with the control group through multiple-regression analyses controlling for age at diagnosis, gender and medication (cDMARDs and corticoids). RESULTS: Established RA patients had significantly increased levels of sphingosine, monohexosylceramide and ceramide compared with controls, when controlling for age and gender. Monohexosylceramide levels remained significantly increased when additionally controlling for medication. On the contrary, SpA patients had significantly decreased levels of ceramide, in both analyses. CONCLUSION: We observed a detectable increase in the levels of certain sphingolipids in the serum of established RA patients when compared with controls, in line with previous observations in the synovial fluid. Such findings provide further evidence that sphingolipids may play a key role in the pathophysiology of RA.

DMS as an orthogonal separation to LC/ESI/MS/MS for quantifying isomeric cerebrosides in plasma and cerebrospinal fluid.

Cerebrosides, including glucosylceramides (GlcCers) and galactosylceramides (GalCers), are important membrane components of animal cells with deficiencies resulting in devastating lysosomal storage disorders. Their quantification is essential for disease diagnosis and a better understanding of disease mechanisms. The simultaneous quantification of GlcCer and GalCer isomers is, however, particularly challenging due to their virtually identical structures. To address this challenge, we developed a new LC/MS-based method using differential ion mobility spectrometry (DMS) capable of rapidly and reproducibly separating and quantifying isomeric cerebrosides in a single run. We show that this LC/ESI/DMS/MS/MS method exhibits robust quantitative performance within an analyte concentration range of 2.8-355 nM. We further report the simultaneous quantification of nine GlcCers (16:0, 18:0, 20:0, 22:0, 23:0, 24:1, 24:0, 25:0, and 26:0) and five GalCers (16:0, 22:0, 23:0, 24:1, and 24:0) molecular species in human plasma, as well as six GalCers (18:0, 22:0, 23:0, 24:1, 24:0 and 25:0) and two GlcCers (24:1 and 24:0) in human cerebrospinal fluid. Our method expands the potential of DMS technology in the field of glycosphingolipid analysis for both biomarker discovery and drug screening by enabling the unambiguous assignment and quantification of cerebroside lipid species in biological samples.

Neutral glycosphingolipids in human blood: a precise mass spectrometry analysis with special reference to lipoprotein-associated Shiga toxin receptors.

Shiga toxin (Stx)-producing Escherichia coli are the leading cause of hemorrhagic colitis and life-threatening extraintestinal complications in humans. Stx1 and Stx2 are transferred by yet to be delineated mechanisms from the intestine to the circulation where they injure microvascular endothelial cells. The resulting vascular lesions cause renal failure and brain damage. Because lipoproteins are potential carriers of Stx through the circulation, we investigated human lipoprotein-associated neutral glycosphingolipids (GSLs) with emphasis on high (globotriaosylceramide) and low (globotetraosylceramide) affinity Stx-receptors. TLC overlay employing Stx1, Stx2, and anti-GSL antibodies demonstrated preferential distribution of globo-series GSLs to very low- and low-density lipoproteins compared with minor association with high-density lipoproteins. Electrospray ionization quadrupole time-of-flight mass spectrometry portrayed C24:0/C24:1 and C16:0 as the major fatty acid of the ceramide moieties of Stx-receptors carrying nonvarying d18:1 sphingosine. This structural heterogeneity was also found in precursor lactosylceramide, glucosylceramide, and galactosylceramide, the last showing an exceptionally high degree of hydroxylated C24 fatty acids. Our findings provide the basis for exploring the functional role of lipoprotein-associated Stx-receptors in human blood.

Enzyme replacement in Fabry's disease, an inborn error of metabolism.

Two patients with Fabry's disease were infused with normal plasma to provide active enzyme (ceramide trihexosidase) for hydrolysis of the plasma substrate, galactosylgalactosylglucosylceramide. Maximum ceramide trihexosidase activity occurred 6 hours after infusion of the plasma, attaining a level approximately 150 percent of that in normal plasma; enzymatic activity was detectable for 7 days. The amount of accumulated substrate in the plasma of these recipients decreased about 50 percent on day 10 after infusion. Thus, periodic replacement of ceramide trihexosidase activity in the plasma of patients with Fabry's disease might lead to consistently lower amounts of substrate in the plasma and a decrease in its rate of accumulation in tissues.

Global glycosphingolipid analysis in urine and plasma of female Fabry disease patients.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase-A, which results in accumulation of the glycosphingolipid (GSL) globotriaosylceramide (Gb3). Gb3 and globotriaosylsphingosine (lyso-Gb3) levels in plasma and urine are used routinely for diagnosis and treatment monitoring. FD female patients are problematic to diagnose and to predict when to begin treatment. Further biomarkers are needed to detect pre-symptomatic females that will develop the chronic symptoms associated with FD. A LC-MS/MS glycosphingolipidomic assay was developed to measure lyso-Gb3 and GSLs from the lysosomal GSL degradation pathway, including globoside (Gb4), Gb3, ceramide dihexosides (CDH) and ceramide monohexosides (CMH). We analysed plasma and urine from a cohort of Fabry patients, grouped according to clinical symptoms and independent of treatment status (asymptomatic females n = 18, symptomatic females n = 18, males n = 27 and control urines n = 16 and control plasmas n = 58). Multivariate and subsequent univariate analysis showed urine GSLs which had highest significance in identifying asymptomatic females were total levels of CDH, in particular the long chain isoforms C22:1,C22:0,C22:1-OH,C22:0-OH,C24:2,C24:0,C24:2-OH,C24:1-OH,C24:0-OH,C26:0 which likely represent Galabiosylceramide (Ga2) and not lactosylceramide. These long chain Ga2 isoforms were found to be 5-fold elevated and more statistically significant (p < 0.0001) than plasma lyso-Gb3 (p < 0.01) in identifying asymptomatic Fabry female patients. Receiver operating characteristic curve analysis gave an area under the curve of 0.82 (p = 0.001) for lyso-Gb3 and 0.88 (p = 0.0006) for long-chain CDH isoforms indicating the long chain CDH isoforms were as, if not more, a better biomarker for the identification of female FD patients.

DEGS1 variant causes neurological disorder.

Sphingolipidoses are monogenic lipid storage diseases caused by variants in enzymes of lipid synthesis and metabolism. We describe an autosomal recessive complex neurological disorder affecting consanguineous kindred. All four affected individuals, born at term following normal pregnancies, had mild to severe intellectual disability, spastic quadriplegia, scoliosis and epilepsy in most, with no dysmorphic features. Brain MRI findings were suggestive of leukodystrophy, with abnormal hyperintense signal in the periventricular perioccipital region and thinning of the body of corpus callosum. Notably, all affected individuals were asymptomatic at early infancy and developed normally until the age of 8-18 months, when deterioration ensued. Homozygosity mapping identified a single 8.7 Mb disease-associated locus on chromosome 1q41-1q42.13 between rs1511695 and rs537250 (two-point LOD score 2.1). Whole exome sequencing, validated through Sanger sequencing, identified within this locus a single disease-associated homozygous variant in DEGS1, encoding C4-dihydroceramide desaturase, an enzyme of the ceramide synthesis pathway. The missense variant, segregating within the family as expected for recessive heredity, affects an evolutionary-conserved amino acid of all isoforms of DEGS1 (c.656A>G, c.764A>G; p.(N219S), p.(N255S)) and was not found in a homozygous state in ExAC and gnomAD databases or in 300 ethnically matched individuals. Lipidomcs analysis of whole blood of affected individuals demonstrated augmented levels of dihydroceramides, dihydrosphingosine, dihydrosphingosine-1-phosphate and dihydrosphingomyelins with reduced levels of ceramide, sphingosine, sphingosine-1-phosphate and monohexosylceramides, as expected in malfunction of C4-dihydroceramide desaturase. Thus, we describe a sphingolipidosis causing a severe regressive neurological disease.

A dialysis procedure for loading erythrocytes with enzymes and lipids.

A dialysis procedure for hypotonic hemolysis has been developed in which erythrocytes can be loaded with water-soluble enzymes, detergent-solubilized enzymes (glucocerebrosidase) and detergent-dispersed glycolipid (glucocerebroside). The procedure allows approx. 40-50% of the added enzyme or glycolipid to be encapsulated. The final intracellular concentration of enzyme or glycolipid is about to the extracellular concentration. The loaded cells can be ingested by macrophage in vitro and the glucocerebroside partially degraded by lysosomal glucocerebrosidase. The use of this procedure for the investogation of Gaucher's disease is discussed.

Lipidomic profiling of plasma and urine from patients with Gaucher disease during enzyme replacement therapy by nanoflow liquid chromatography-tandem mass spectrometry.

Gaucher disease (GD) is a rare genetic disorder that arises from lipid species, especially monohexosylceramide (MHC), accumulating in different organs. GD results from a ß-glucocerebrosidase deficiency, causing metabolic or neurologic complications. This study comprehensively profiled lipids from patients and healthy controls to discover active lipid species related to GD. Most studies have evaluated lipids from one type of biological sample, such as plasma, urine, or spinal fluid, which are the main sources of lipids in human bodies. The purpose of this study, however, was to collect and assess both plasma and urine samples from a group of individuals, explore the lipids, and select characteristic species that show significant differences between controls and patients from the two sources. Also, the response of lipids to enzyme replacement therapy (ERT), which is targeted to reduce excessive lipid accumulation within lysosomes, was investigated by obtaining plasma and urine from patients after receiving the therapy. Most lipid species were found in both plasma and urine but their concentrations differed, and some species were found in either plasma or urine only. Out of 125 plasma and 105 urinary lipids that were identified by nLC-ESI-MS/MS, 20 plasma and 10 urinary lipids were selected as characteristic species for having average concentrations that were significantly increased or decreased in patients by greater than 2-fold. Moreover, the concentrations of most lipids that showed greater than 2-fold of difference in patients decreased after ERT indicating that these species were directly or indirectly affected by the therapy.

Existence of glucosaminyl lactosyl ceramide (amino CTH-I) in human erythrocyte membranes as a possible precursor of blood group-active glycolipids.

A novel glycolipid containing N-acetylglucosamine (lacto-N-triose II ceramide, Amino CTH-I) was isolated as a minor component from the ceramide trihexoside fraction of human erythrocyte lipids. This glycolipid was purified by column chromatography and preparative high-speed liquid chromatography on Iatrobeads. Its structure was found to be N-acetylglucosaminyl(beta 1-3)galactosyl(beta 1-4)glucosyl-(beta 1-1)ceramide, so it is suggested that it may be a precursor of paragloboside and blood group-active glycolipids.

Heterophile antibodies to rabbit erythrocytes in human sera and identification of the antigen as a glycolipid.

Normal human sera contain heterophile hemagglutinins to rabbit erythrocytes which are different from anti-B isoantibody and other heterophile antibodies such as Hanganutziu-Deicher antibody or Paul-Bunnell antibody. The antigen to this antibody was purified from rabbit erythrocyte stroma, and identified as pentaglycosyl ceramide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer.

Accumulation of lactosyl ceramide in leukocytes of patients with adult Gaucher's disease.

Glycosphingolipids were isolated from the leukocytes of 10 patients with Type I, chronic nonneuronopathic (adult) Gaucher's disease and 12 normal subjects, by silicic acid column chromatography and thin-layer chromatography. Quantitation of the individual glycosyl ceramides was achieved by the determination of hexose and sphingosine content, using colorimetric and fluorometric procedures. Lactosyl ceramide, which is the main glycolipid in leukocytes of normal subjects, was significantly increased in the leukocytes of patients with Gaucher's disease. On the other hand, the amount of glycosyl ceramide, which is the main glycolipid accumulating in the reticuloendothelial cells of patients with Gaucher's disease, was similar in Gaucher and in control leukocytes.

Distribution of antithrombin III and glucosylceramide in human plasma lipoproteins and lipoprotein deficient plasma.

We have investigated the distribution of antithrombin-III and glucosylceramide (Glc-Cer) in human plasma, plasma lipoproteins and lipoprotein-deficient plasma. Antithrombin III activity was measured employing immunochemical and biological assays. Glc-Cer was quantified by gas liquid chromatography (GLC). Whole plasma contained 145 micrograms antithrombin III/ml plasma, all of which was associated with the lipoprotein-deficient plasma (d greater than 1.25 g/ml). Whereas, most if not all the plasma GlcCer was associated with plasma low density lipoproteins (LDL) (d-1.022-1.055 g/ml) and high density lipoproteins (HDL) (d-1.063-1.25). GlcCer was not found in the lipoprotein-deficient plasma. We conclude that GlcCer on lipoproteins does not contribute to antithrombin III activity. Moreover, the absence of GlcCer in lipoprotein-deficient plasma does not impair antithrombin-III activity.

Plasma cerebrosides in stroke and multiple sclerosis.

This investigation was conducted to determine whether or not plasma galactosyl ceramides were elevated in patients with stroke and multiple sclerosis, and to determine glycosyl ceramide concentrations in older, normal subjects. It was hypothesized that central nervous system destruction, like that which occurs in stroke or in the demyelination characteristic of multiple sclerosis, might be reflected by changes in plasma glycosyl ceramides, specifically by an increased percentage of galactosyl ceramide. Glycosyl ceramides were analyzed in duplicate 10 ml aliquots of plasma from each of seven patients with stroke, five patients with multiple sclerosis and five control subjects age-matched with stroke patients. Mean percentages of galactosyl ceramide for both controls (11.06 percent) and multiple sclerosis (11.40 percent) were strikingly similar. The percent of galactosyl ceramides for stroke was slightly elevated (14.32 percent) but there were no significant differences at the p=greater than 0.05 level.

Plasma ceramide and glucosylceramide metabolism is altered in sporadic Parkinson's disease and associated with cognitive impairment: a pilot study.

BACKGROUND: Mutations in the gene coding for glucocerebrosidase (GBA), which metabolizes glucosylceramide (a monohexosylceramide) into glucose and ceramide, is the most common genetic risk factor for sporadic Parkinson's disease (PD). GBA mutation carriers are more likely to have an earlier age of onset and to develop cognitive impairment and dementia. We hypothesized that plasma levels of lipids involved in ceramide metabolism would also be altered in PD non-GBA mutation carriers and associated with worse cognition. METHODS: Plasma ceramide, monohexosylceramide, and lactosylceramide levels in 26 cognitively normal PD patients, 26 PD patients with cognitive impairment or dementia, and 5 cognitively normal non-PD controls were determined by LC/ESI/MS/MS. RESULTS: Levels of all lipid species were higher in PD patients versus controls. Among PD patients, levels of ceramide C16:0, C18:0, C20:0, C22:0, and C24:1 and monohexosylceramide C16:0, C20:0 and C24:0 species were higher (all P<0.05) in those with versus without cognitive impairment. CONCLUSION: These results suggest that plasma ceramide and monohexosylceramide metabolism is altered in PD non-GBA mutation carriers and that higher levels are associated with worse cognition. Additional studies with larger sample sizes, including cognitively normal controls, are needed to confirm these findings.