Serum Sphingolipidomic Analysis in Acne Vulgaris Patients.

BACKGROUND: Limited data are available on serum levels of different sphingomyelin (SM) and ceramide (CER) species in acne vulgaris (AV). OBJECTIVES: This study aimed to identify circulating levels of neutral sphingomyelinase activity (N-SMase), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), C16-C24 SMs and C16-C24 CERs in AV patients and controls. MATERIAL AND METHODS: Serum was collected from 30 AV patients and 20 age, gender-matched control subjects. Serum levels of C16-C24 SMs and C16-C24 CERs were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of N-SMase was assayed by standard kit methods, C1P and S1P levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: A significant increase was observed in serum levels of C16 SM in patients with AV compared to controls. No significant difference was found in C18 and C24 SM levels between the two groups. Very-long-chain C24 CER was significantly decreased in AV patients compared to controls. Long chain C16-C20 CER levels showed no significant difference between AV patients and controls. A significant positive correlation was found between serum total cholesterol levels and all measured SMs and CERs in both the control and patient groups. Patients with AV had increased circulating levels of C16 SM, C1P and lower circulating levels of C24 CER compared to healthy controls, which may provide prognostic value for the disease. CONCLUSIONS: Future studies are needed to understand the role of altered sphingolipid levels in the pathophysiology of AV.

Diclofenac induced gastrointestinal and renal toxicity is alleviated by thymoquinone treatment.

The aim of this study was to investigate whether thymoquinone (TQ) could alleviate diclofenac (DCLF)-induced gastrointestinal and renal toxicity in rats. Diclofenac was administered via intramuscular injection twice daily for 5 days and TQ was given by gavage for the same period. Hematological and biochemical profiles were measured with autoanalyzers while reactive oxygen/nitrogen species (ROS/RNS) generation and total antioxidant capacity (TAC) were assayed by standard kits. Tissue injuries were evaluated by microscopy and histopathological scoring. Diclofenac treatment caused kidney and liver function test abnormalities, reduced hematocrit and hemoglobin levels but increased WBC and platelet counts. Histopathological findings showed renal tubular damage, gastrointestinal lesions and increased fibrosis in DCLF treated rats. Thymoquinone administration, along with DCLF treatment, attenuated hematological test abnormalities and DCLF induced renal functional impairment as evident by significantly restored serum creatinine and blood urea nitrogen levels. Similarly, TQ treatment significantly alleviated liver function test abnormalities and decreased tissue injury in the stomach and duodenum. Diclofenac treatment caused increased ROS/RNS formation and decreased TAC in the kidney, stomach and duodenal tissue. Thymoquinone administration increased gastrointestinal and renal TAC in DCLF treated rats. These results indicate that TQ could ameliorate gastrointestinal and renal toxicity induced by high dose DCLF treatment.

Decreased Serum Levels of Sphingomyelins and Ceramides in Sickle Cell Disease Patients.

Limited data are available on the serum levels of different sphingomyelin (CerPCho) and ceramide (CER) species in sickle-cell disease (SCD). This study was aimed at identifying the levels of C16-C24 CerPCho and C16-C24 CER in serum obtained from SCD patients and controls. Circulating levels of neutral sphingomyelinase (N-SMase) activity, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P) were also determined. Blood was collected from 35 hemoglobin (Hb)A volunteers and 45 homozygous HbSS patients. Serum levels of C16-C24 CerPCho and C16-C24 CER were determined by an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of N-SMase was assayed by standard kit methods, and C1P and S1P levels were determined by enzyme-linked immunosorbent assay. A significant decrease was observed in the serum levels of C18-C24 CerPCho in patients with SCD compared to controls. No significant difference was found in C16 CerPCho levels between the two groups. Very-long-chain C22-C24 CER were significantly decreased in SCD, while long-chain C16-C20 CER levels showed no significant difference between SCD patients and controls. Significant positive correlation was found between the serum total cholesterol levels and C18-C24 CerPCho and C22-C24 CER in SCD patients. Patients with SCD had significantly elevated serum activity of N-SMase as well as increased circulating levels of C1P and S1P compared to controls. The decrease in serum levels of C18-C24 CerPCho in patients with SCD was accompanied by decreased levels of C22-C24 CER. Future studies are needed to understand the role of decreased CerPCho and CER in the pathophysiology of SCD.

Neutral sphingomyelinase inhibition alleviates apoptosis, but not ER stress, in liver ischemia-reperfusion injury.

Previous studies have revealed the activation of neutral sphingomyelinase (N-SMase)/ceramide pathway in hepatic tissue following warm liver ischemia reperfusion (IR) injury. Excessive ceramide accumulation is known to potentiate apoptotic stimuli and a link between apoptosis and endoplasmic reticulum (ER) stress has been established in hepatic IR injury. Thus, this study determined the role of selective N-SMase inhibition on ER stress and apoptotic markers in a rat model of liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reactions monitoring (MRM) method using ultrafast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared with controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78 kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c, and caspase-3, -8, and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.

Decreased eicosapentaenoic acid levels in acne vulgaris reveals the presence of a proinflammatory state.

This study aimed to determine circulating levels of polyunsaturated fatty acids (PUFAs), secretory phospholipase A2 (sPLA2), lipoprotein lipase (LPL) and measure circulating protein levels of angiopoietin-like protein 3 (ANGPTL3), ANGPTL4, cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in patients with acne vulgaris. Serum from 21 control subjects and 31 acne vulgaris patients were evaluated for levels of arachidonic acid (AA, C20:4n- 6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). PUFA levels were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods Serum EPA levels were significantly decreased while AA/EPA and DGLA/EPA ratio were significantly increased in acne vulgaris patients compared to controls. Serum levels of AA, DGLA and DHA showed no significant difference while activity of sPLA2 and LPL were significantly increased in acne vulgaris compared to controls. Results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly decreased serum EPA levels and increased activity of sPLA2, AA/EPA and DGLA/EPA ratio. Increased LPL activity in the serum of acne vulgaris patients can be protective through its anti-dyslipidemic actions. This is the first study reporting altered EPA levels and increased sPLA2 activity in acne vulgaris and supports the use of omega-3 fatty acids as adjuvant treatment for acne patients.

Inhibition of neutral sphingomyelinase decreases elevated levels of nitrative and oxidative stress markers in liver ischemia-reperfusion injury.

Oxidative stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS)-2 have been shown in the pathogenesis of liver ischemia-reperfusion (IR) injury. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression therefore this study determined the role of selective N-SMase inhibition on nitrative and oxidative stress markers following liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Nitrative and oxidative stress markers were determined by evaluating NOS2 expression, protein nitration, nitrite/nitrate levels, 4-hydroxynonenal (HNE) formation, protein carbonyl levels and xanthine oxidase/xanthine dehydrogenase (XO/XDH) activity. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reaction monitoring method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared to controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. NOS2 expression, nitrite/nitrate levels and protein nitration were significantly greater in IR injury and decreased with N-SMase inhibition. Treatment with a selective N-SMase inhibitor significantly decreased HNE formation, protein carbonyl levels and the hepatic conversion of XO. Data confirm the role of nitrative and oxidative injury in IR and highlight the protective effect of selective N-SMase inhibition. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate oxidative injury in liver I/R injury.

Pentoxifylline Alleviates Early Brain Injury in a Rat Model of Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease frequently caused by ruptured aneurysms. Early brain injury (EBI) is the primary cause of morbidity and mortality in patients diagnosed with SAH and is associated with increased intracranial pressure, decreased cerebral blood flow and cerebral ischemia. Pentoxifylline (PTX) is a methylxanthine derivative clinically proven to improve perfusion in the peripheral microcirculation and has been shown to have neuroprotective effects in brain trauma and global cerebral ischemia in experimental animal models. This study aimed to determine the effect of PTX in experimental SAH, which has not been investigated yet. METHODS: An experimental SAH model was induced in male Wistar rats by autologous blood injection into the prechiasmatic cistern, and PTX was injected intraperitoneally immediately after SAH. The effects of PTX were evaluated 24 h after SAH via assessing the cerebral ultrastructure via transmission electron microscopy (TEM). Brain edema, blood-brain barrier (BBB) permeability, red blood cell deformability, tumor necrosis factor-alpha (TNF-alpha), nitrite-nitrate levels and apoptotic neuron death were also determined 24 h after SAH. The BBB permeability was measured by Evans blue (EB) extravasation, erythrocyte deformability was determined by filtration technique, and TNF-alpha and reactive nitrogen metobolites were analyzed in brain tissue by ELISA and spectral analysis, respectively. Apoptotic neurons were determined in brain sections by cleaved caspase-3 immunohistochemical analysis, and expression intensity was quantified using image J software. RESULTS: Cerebral ultrastructure in SAH group animals revealed intense perivascular edema and distortion in the astrocyte foot processes. PTX treatment attenuated structural deterioration due to SAH. Brain water content, BBB permeability, TNF-alpha, nitrite-nitrate levels and apoptotic neuronal death were significantly increased 24 h after SAH and were significantly alleviated by PTX treatment. There was no significant change in red cell deformability after SAH. CONCLUSIONS: Our results show that PTX reduces brain edema, BBB permeability, TNF-alpha expression, reactive nitrogen metobolites and apopotosis in experimental SAH. Based on our findings we suggest that PTX exerts neuroprotection against SAH-induced EBI, which might be associated with the inhibition of inflammation and apoptotic neuronal cell death.

Mass spectrometric quantification of urinary human liver fatty acid binding protein in renal transplant recipients.

RATIONALE: Urinary liver fatty acid binding protein (L-FABP) has been evaluated as a promising early biomarker of renal ischemia in human kidney transplant patients. The use of L-FABP in clinical practice requires that this biomarker be associated with an analytical method that combines specificity, accuracy and robustness. This study aimed to evaluate an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography coupled with tandem mass spectrometry to measure urinary L-FABP levels in renal transplant recipients. METHODS: Purified recombinant human L-FABP tryptic standard was analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS/MS) and liquid chromatography (LC)/MS/MS to select for peptides that provided specificity and adequate response in developing an MRM method for urinary L-FABP quantification. Human urine samples collected from kidney transplant recipients were isolated, concentrated, precipitated and trypsin digested before mass spectrometric analysis of L-FABP. L-FABP levels were also measured in urine samples by enzyme immunoassay. RESULTS: The tryptic peptide ion MH(+) of (50) FTITAGSK(57) (m/z 824) provided an adequate signal and was used for quantification of L-FABP under conditions employed for LC/MS/MS analysis. MALDI-TOF-MS/MS spectra obtained by collision-induced dissociation of the parent MH(+) ion (50) FTITAGSK(57) resulted in a y3 product ion that was used for quantitative analysis by the MRM method. Urinary L-FABP content measured by both ELISA and LC/MS/MS after transplantation was significantly higher compared to before transplantation levels. The Spearman correlation coefficient between the two methods was statistically significant. Intra-day and inter-day coefficients of variation provided good repeatability and reproducibility for validation of LC/MS/MS analysis. CONCLUSIONS: LC/MS/MS quantification of L-FABP may provide a new reference method to determine changes in this potential biomarker in human kidney transplant patients.

Analysis of polyunsaturated fatty acids and the omega-6 inflammatory pathway in hepatic ischemia/re-perfusion injury.

The aim of the present study was to assess omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in liver tissue and evaluate changes in the n­6-associated inflammatory pathway following liver ischemia/re­perfusion (IR) injury. Male Wistar rats which were allowed free access to standard rat chow were included in the study. Blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60 min, followed by 60 min of re­perfusion. Levels of arachidonic acid (AA, C20:4n­6), dihomo­gamma­linolenic acid (DGLA, C20:3n­6), eicosapentaenoic acid (EPA, C20:5n­3) and docosahexaenoic acid (DHA, C22:6n­3) in liver tissue were determined by an optimized multiple reaction monitoring method using ultra fast­liquid chromatography coupled with tandem mass spectrometry. Phospholipase A2 (PLA2), cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in the n­6 inflammatory pathway. Total histopathological score of cellular damage were significantly increased following hepatic IR injury. n­3 and n­6 PUFA levels were significantly increased in post­ischemic liver tissue compared to those in non­ischemic controls. No significant difference was observed in the AA/DHA and AA/EPA ratio in post­ischemic liver tissues compared with that in the control. Tissue activity of PLA2 and COX as well as PGE2 levels were significantly increased in post­ischemic liver tissues compared to those in non­ischemic controls. The results of the present study suggested that increased hydrolysis of fatty acids via PLA2 triggers the activity of COX and leads to increased PGE2 levels. Future studies evaluating agents which block the formation of eicosanoids derived from n­6 PUFAs may facilitate the development and application of treatment strategies in liver injury following IR.

Inhibition of neutral sphingomyelinase decreases arachidonic acid mediated inflammation in liver ischemia-reperfusion injury.

This study aimed to determine the role of selective neutral sphingomyelinase (N-SMase) inhibition on arachidonic acid (AA) mediated inflammation following liver ischemia-reperfusion (IR) injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of AA in liver tissue were determined by multiple reaction monitoring (MRM) using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Phospholipase A2 (PLA2), cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in liver tissue. Arachidonic acid levels, activity of PLA2, COX and PGE2 levels were significantly increased in postischemic liver tissue compared to nonischemic controls. N-SMase inhibition significantly decreased COX activity and PGE2 levels in postischemic liver. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate inflammation in liver I/R injury.