High level of phosphatidylcholines/lysophosphatidylcholine ratio in urine is associated with prostate cancer.

The altered levels of phospholipids (PLs) and lysophospholipids (LPLs) in prostate cancer (CaP) and benign tissues in our previous findings prompted us to explore PLs and LPLs as potential biomarkers for CaP. Urinary lipidomics has attracted increasing attention in clinical diagnostics and prognostics for CaP. In this study, 31 prostate tissues obtained from radical prostatectomy were assessed using high-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS). Urine samples were collected after digital rectal examination (DRE), and urinary lipids were extracted using the acidified Bligh-Dyer method. The discovery set comprised 75 patients with CaP and 44 with benign prostatic hyperplasia (BPH) at Kyoto University Hospital; the validation set comprised 74 patients with CaP and 59 with BPH at Osaka University Hospital. Urinary lipidomic screening was performed using MALDI time-of-flight MS (MALDI-TOF/MS). The levels of urinary lysophosphatidylcholine (LPC) and phosphatidylcholines (PCs) were compared between the CaP and BPH groups. The (PC [34:2] + PC [34:1])/LPC (16:0) ratio was significantly higher (P < .001) in CaP tissues than in benign epithelial tissues. The urinary PCs/LPC ratio was significantly higher (P < .001) in the CaP group than in the BPH group in the discovery and validation sets.

Qualified method for the estimation of di-18:1 bis(monoacylglycero)phosphate in urine, a noninvasive biomarker to monitor drug-induced phospholipidosis.

Aim: Our objective was to develop and qualify a bioanalytical method for the estimation of di-18:1-bis(monoacylglycero)phosphate (di-18:1 BMP) as a urinary biomarker for the assessment of drug-induced phospholipidosis and demonstrate its application in a preclinical study. Methodology/results: di-18:1 BMP was extracted by liquid-liquid extraction using n-butanol and analyzed by LC-MS/MS. The qualified method was selective, precise, robust and accurate across the linearity range (0.2-250 ng/ml). Qualified method was then used to assess chloroquine-induced phospholipidosis in rats dosed at 120 mg/kg for 5 days. A fivefold increase in di-18:1 BMP was observed on Day 5 compared with predose. Conclusion: Di-18:1 BMP can be used as a noninvasive biomarker to assess/screen compounds that could cause drug-induced phospholipidosis in rats.

Comparative evaluation of the extraction and analysis of urinary phospholipids and lysophospholipids using MALDI-TOF/MS.

Lipids, particularly phospholipids (PLs) and lysophospholipids (LPLs), are attracting increasing scientific interest for their biological functions in cells and their potential as disease biomarkers for Alzheimer's disease and several types of cancer. Urinary PLs and LPLs could be ideal clinical biomarkers, because urine can be collected easily and noninvasively. However, due to their very low concentrations in urine compared with the relatively large quantity of contaminants in this matrix, efficient extraction and sensitive detection are required for analyzing urinary PLs and LPLs. In this study, various methods for analyzing PLs and LPLs in urine were compared and optimized from a clinical perspective. An optimized lipid extraction method and a matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) were established using two external ionization standards and an internal standard mix containing 13 human urinary lipids. 9-Aminoacridine (9-AA) was a useful and effective matrix for the MALDI-TOF/MS analysis of all the internal standard lipids in both positive and negative ion modes. However, it was necessary to determine the proportional lipid concentrations from the balance between the extracted lipid and the matrix. The extraction efficiency and reproducibility of the acidified Bligh and Dyer method were excellent for both positively and negatively charged lipids. Analysis of small volumes of urine was the most efficient with the 9-AA MALDI matrix at concentrations of or below 5 mM. The combined analytical procedures allowed rapid and comprehensive screening of low concentrations of PLs and LPLs in clinical samples.

Urinary lysophopholipids are increased in diabetic patients with nephropathy.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease that frequently leads to end stage renal failure. Lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are lysophospholipid mediators shown to accumulate in kidney and to promote renal inflammation and tubulo-interstitial fibrosis in diabetic rodent models. Here we assessed whether LPA and LPC were associated to the development of nephropathy in diabetic human patients. Several molecular species of LPA and LPC were quantified by LC/MS-MS in urine and plasma from type 2 diabetic patients with (cases; n=41) or without (controls, n=41) nephropathy symptoms (micro/macro-albuminuria and eGFR<60ml/min/1.73m2). Cases and controls were matched for sex, age and diabetes duration. Six species were detected in urine for both LPA and LPC, LPA16:0, LPA20:4, LPC16:0, LPC18:0, LPC18:1, and LPC18:2 that were significantly more concentrated in cases than in controls. Total LPC and LPA (sum of detected species) were significantly and exclusively associated with albuminuria (P<0.0001 and P=0.0009 respectively) and were significantly higher in the 3rd when compared to the 1st albuminuria tertile in cases. Plasma lysophospholipids showed a different species profile urine and their concentrations were not different between cases and controls. In conclusion, urine concentration of lysophospholipids increases in diabetic patients with DN as the likely result of their co-excretion with albumin combined with possible local production by kidney. Because LPA and LPC are known to promote renal inflammation and tubulo-interstitial fibrosis, their increased production in DN could participate to the development of kidney damage associated with diabetes.

Increased urinary lysophosphatidic acid in mouse with subtotal nephrectomy: potential involvement in chronic kidney disease

Increased incidence of chronic kidney disease (CKD) with consecutive progression to end-stage renal disease represents a significant burden to healthcare systems. Renal tubulointerstitial fibrosis (TIF) is a classical hallmark of CKD and is well correlated with the loss of renal function. The bioactive lysophospholipid lysophosphatidic acid (LPA), acting through specific G-protein-coupled receptors, was previously shown to be involved in TIF development in a mouse model of unilateral ureteral obstruction. Here, we study the role of LPA in a mouse subjected to subtotal nephrectomy (SNx), a more chronic and progressive model of CKD. Five months after surgical nephron reduction, SNx mice showed massive albuminuria, extensive TIF, and glomerular hypertrophy when compared to sham-operated animals. Urinary and plasma levels of LPA were analyzed using liquid chromatography tandem mass spectrometry. LPA was significantly increased in SNx urine, not in plasma, and was significantly correlated with albuminuria and TIF. Moreover, SNx mice showed significant downregulation in the renal expression of lipid phosphate phosphohydrolases (LPP1, 2, and 3) that might be involved in reduced LPA bioavailability through dephosphorylation. We concluded that SNx increases urinary LPA through a mechanism that could involve co-excretion of plasma LPA with albumin associated with a reduction of its catabolism in the kidney. Because of the previously demonstrated profibrotic activity of LPA, the association of urinary LPA with TIF suggests the potential involvement of LPA in the development of advanced CKD in the SNx mouse model. Targeting LPA metabolism might represent an interesting approach in CKD treatment (AU)

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Increased urinary lysophosphatidic acid in mouse with subtotal nephrectomy: potential involvement in chronic kidney disease.

Increased incidence of chronic kidney disease (CKD) with consecutive progression to end-stage renal disease represents a significant burden to healthcare systems. Renal tubulointerstitial fibrosis (TIF) is a classical hallmark of CKD and is well correlated with the loss of renal function. The bioactive lysophospholipid lysophosphatidic acid (LPA), acting through specific G-protein-coupled receptors, was previously shown to be involved in TIF development in a mouse model of unilateral ureteral obstruction. Here, we study the role of LPA in a mouse subjected to subtotal nephrectomy (SNx), a more chronic and progressive model of CKD. Five months after surgical nephron reduction, SNx mice showed massive albuminuria, extensive TIF, and glomerular hypertrophy when compared to sham-operated animals. Urinary and plasma levels of LPA were analyzed using liquid chromatography tandem mass spectrometry. LPA was significantly increased in SNx urine, not in plasma, and was significantly correlated with albuminuria and TIF. Moreover, SNx mice showed significant downregulation in the renal expression of lipid phosphate phosphohydrolases (LPP1, 2, and 3) that might be involved in reduced LPA bioavailability through dephosphorylation. We concluded that SNx increases urinary LPA through a mechanism that could involve co-excretion of plasma LPA with albumin associated with a reduction of its catabolism in the kidney. Because of the previously demonstrated profibrotic activity of LPA, the association of urinary LPA with TIF suggests the potential involvement of LPA in the development of advanced CKD in the SNx mouse model. Targeting LPA metabolism might represent an interesting approach in CKD treatment.

Toluene diisocyanate: Induction of the autotaxin-lysophosphatidic acid axis and its association with airways symptoms.

Diisocyanates are industrial chemicals which have a wide range of applications in developed and developing countries. They are notorious lung toxicants and respiratory sensitizers. However, the mechanisms behind their adverse effects are not adequately characterized. Autotaxin (ATX) is an enzyme producing lysophosphatidic acid (LPA), and the ATX-LPA axis has been implicated in lung related inflammatory conditions and diseases, including allergic asthma, but not to toxicity of environmental low-molecular-weight chemicals. We investigated effects of toluene diisocyanate (TDI) on ATX induction in human lung epithelial cell models, and we correlated LPA-levels in plasma to biomarkers of TDI exposure in urine collected from workers exposed to <5ppb (parts per billion). Information on workers' symptoms was collected through interviews. One nanomolar TDI robustly induced ATX release within 10min in vitro. A P2X7- and P2X4-dependent microvesicle formation was implicated in a rapid ATX release and a subsequent protein synthesis. Co-localization between purinergic receptors and ATX was documented by immunofluorescence and confocal microscopy. The release was modulated by monocyte chemoattractant protein-1 (MCP-1) and by extracellular ATP. In workers, we found a dose-response relationship between TDI exposure biomarkers in urine and LPA levels in plasma. Among symptomatic workers reporting "sneezing", the LPA levels were higher than among non-symptomatic workers. This is the first report indicating induction of the ATX-LPA axis by an environmental low-molecular-weight chemical, and our data suggest a role for the ATX-LPA axis in TDI toxicity.

Di-22:6-bis(monoacylglycerol)phosphate: A clinical biomarker of drug-induced phospholipidosis for drug development and safety assessment.

The inability to routinely monitor drug-induced phospholipidosis (DIPL) presents a challenge in pharmaceutical drug development and in the clinic. Several nonclinical studies have shown di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP) to be a reliable biomarker of tissue DIPL that can be monitored in the plasma/serum and urine. The aim of this study was to show the relevance of di-22:6-BMP as a DIPL biomarker for drug development and safety assessment in humans. DIPL shares many similarities with the inherited lysosomal storage disorder Niemann-Pick type C (NPC) disease. DIPL and NPC result in similar changes in lysosomal function and cholesterol status that lead to the accumulation of multi-lamellar bodies (myeloid bodies) in cells and tissues. To validate di-22:6-BMP as a biomarker of DIPL for clinical studies, NPC patients and healthy donors were classified by receiver operator curve analysis based on urinary di-22:6-BMP concentrations. By showing 96.7-specificity and 100-sensitivity to identify NPC disease, di-22:6-BMP can be used to assess DIPL in human studies. The mean concentration of di-22:6-BMP in the urine of NPC patients was 51.4-fold (p ≤ 0.05) above the healthy baseline range. Additionally, baseline levels of di-22:6-BMP were assessed in healthy non-medicated laboratory animals (rats, mice, dogs, and monkeys) and human subjects to define normal reference ranges for nonclinical/clinical studies. The baseline ranges of di-22:6-BMP in the plasma, serum, and urine of humans and laboratory animals were species dependent. The results of this study support the role of di-22:6-BMP as a biomarker of DIPL for pharmaceutical drug development and health care settings.

Comparison of urinary and serum levels of di-22:6-bis(monoacylglycerol)phosphate as noninvasive biomarkers of phospholipidosis in rats.

Phospholipidosis (PLD), an abnormal accumulation of phospholipids within tissues, is observed during the preclinical testing of many pharmaceutical drugs. Diagnosis of PLD is currently based on morphologic criteria. An understanding of the clinical incidence of PLD and its possible relationship to adverse drug reactions has been hampered by the absence of noninvasive biomarkers for PLD. The uncommon phospholipid di-docosahexaenoyl bis(monoacylglycerol) phosphate (di-22:6-BMP) has been proposed as a potential urinary biomarker for PLD, but data on the utility of serum di-22:6-BMP measurements in diagnosing PLD is more limited. In this report, we compared the performance of serum and urinary di-22:6-BMP as biomarkers for PLD in rats treated with the PLD-inducing drugs amiodarone and 4,4'-diethylaminoethoxyhexestrol dihydrochloride or the hepatotoxicant acetaminophen (APAP). Serum levels of di-22:6-BMP showed a higher correlation to a generalized PLD incidence score than did levels in urine, but were unexpectedly elevated in rats with marked levels of APAP-induced liver necrosis. When samples were filtered based on serum ALT or liver histopathology thresholds, the diagnostic accuracy of serum di-22:6-BMP for PLD improved to the high level observed for urinary di-22:6-BMP without sample exclusion. These data help define the potential context-of-use of serum di-22:6-BMP as a non-clinical biomarker of PLD.

Increased serum concentration of sphingosine-1-phosphate in juvenile-onset systemic lupus erythematosus.

PURPOSE: Sphingosine-1-phosphate (S1P) is an active sphingolipid with chemotactic abilities and has been linked to inflammatory mediators and autoimmune disease. The aim of this study was to assess whether children with juvenile-onset systemic lupus erythematosus (JSLE) express increased systemic and/or urinary concentrations of S1P. METHODS: A subgroup of patients participating in the UK JSLE Cohort Study, were invited to participate. Cross sectional serum and urine samples were prospectively collected along with demographic and standard clinical data. Results were compared to a cohort of disease controls (Henoch Schonlein Purpura; HSP) and healthy controls (HC). RESULTS: The median age of JSLE patients (n = 15) was 13.6 years (7.2-16.9 years). The serum concentrations of S1P in JSLE patients (7.4 uM, IQR 6.3-12.3 uM) were statistically significantly increased when compared to patients with HSP (n = 10; 5.2 uM, IQR 4.0-7.9 uM; p = 0.016) and HCs (n = 10; 3.8 uM, IQR 2.1-5.8 uM; p = 0.003). There was a trend towards increased serum S1P concentrations between patients with active lupus nephritis (n = 8; 8.7 uM, IQR 6.2-15.3 uM) compared to lupus non-nephritis (n = 7; 6.6 uM, IQR 6.3-10.6 uM; p = 0.355). No relationship was found between disease activity markers and S1P. Urine S1P concentrations were no different between JSLE patients (56.0 nM, IQR 40.3-96.6 nM) and HCs (58.7 nM, IQR 0-241.9 nM; p = 0.889). CONCLUSIONS: We have demonstrated, for the first time, an increased serum concentration of S1P in a cohort of JSLE patients. These findings highlight a role of S1P in the pathophysiology of JSLE that warrants further investigation.

Phospholipidosis in rats treated with amiodarone: serum biochemistry and whole genome micro-array analysis supporting the lipid traffic jam hypothesis and the subsequent rise of the biomarker BMP.

To provide mechanistic insight in the induction of phospholipidosis and the appearance of the proposed biomarker di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP), rats were treated with 150 mg/kg amiodarone for 12 consecutive days and analyzed at three different time points (day 4, 9, and 12). Biochemical analysis of the serum revealed a significant increase in cholesterol and phospholipids at the three time points. Bio-analysis on the serum and urine detected a time-dependent increase in BMP, as high as 10-fold compared to vehicle-treated animals on day 12. Paralleling these increases, micro-array analysis on the liver of treated rats identified cholesterol biosynthesis and glycerophospholipid metabolism as highly modulated pathways. This modulation indicates that during phospholipidosis-induction interactions take place between the cationic amphiphilic drug and phospholipids at the level of BMP-rich internal membranes of endosomes, impeding cholesterol sorting and leading to an accumulation of internal membranes, converting into multilamellar bodies. This process shows analogy to Niemann-Pick disease type C (NPC). Whereas the NPC-induced lipid traffic jam is situated at the cholesterol sorting proteins NPC1 and NPC2, the amiodarone-induced traffic jam is thought to be located at the BMP level, demonstrating its role in the mechanism of phospholipidosis-induction and its significance for use as a biomarker.

Comparison of the diagnostic accuracy of di-22:6-bis(monoacylglycerol)phosphate and other urinary phospholipids for drug-induced phospholipidosis or tissue injury in the rat.

Cationic amphiphilic drugs and aminoglycoside antibiotics can induce phospholipidosis (PLD), an abnormal accumulation of phospholipids in lysosome-derived vesicles, in preclinical studies. The incidence of PLD in patients and its clinical relevance are difficult to assess without noninvasive biomarkers. Di-docosahexaenoyl bis(monoacylglycerol)phosphate (di-22:6-BMP) is a phospholipid that is enriched in lysosomal membranes and a proposed urinary biomarker of drug-induced PLD. The specificity of di-22:6-BMP for PLD was compared to other phospholipid species that can increase in urine with nephrotoxicity. Using liquid chromatography coupled to mass spectrometry, 12 phospholipids were assayed in the urine of rats treated with drugs that induced PLD or caused renal or skeletal muscle injury. In receiver operating curve analyses, urinary di-22:6-BMP was a significantly better predictor of PLD and the least predictive of tissue injury of the phospholipids assayed. The data provide evidence supporting the use of di-22:6-BMP as a urinary biomarker of PLD in rats.

Targeted analysis of sphingoid precursors in human biofluids by solid-phase extraction with in situ derivatization prior to µ-LC-LIF determination.

A method for determination of two relevant sphingoid precursors such as sphingosine and sphinganine and the corresponding conjugates sphingosine 1-phosphate and sphinganine 1-phosphate in human urine and serum is here presented. The method is characterized by a solid- phase extraction step with in situ derivatization of the sphingolipids in the eluate (o-phthaldialdehyde derivatives) to obtain fluorescent compounds. In this way, sample preparation was completely performed in a single automated step by means of a lab-on-valve system. Derivatized analytes were injected into a liquid chromatography system operating at micro regime and detected by laser-induced fluorescence. For determination of sphingoid phosphates, they were enzymatically converted to free sphingoids to obtain stable fluorescent derivatives. The detection limits were in the range 4.2-10.2 ng mL(-1) for serum and 0.56-1.36 ng mL(-1) for urine, with repeatability ranging from 3.9% to 6.2% expressed as relative standard deviation. The method was validated by direct infusion tandem mass spectrometry in multiple reaction monitoring to compare results provided by analysis of biofluids and to confirm the identity of the target compounds. Sensitivity and precision were better than or similar to those provided by the confirmatory method. The automation of sample preparation enables to scale-down this step and improves precision by minimization of human intervention, being thus suitable for clinical analysis.

Biomarkers to monitor drug-induced phospholipidosis.

Di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) was identified as a promising phospholipidosis (PL) biomarker in rats treated with either amiodarone, gentamicin, or azithromycin. Sprague-Dawley rats received either amiodarone (150 mg/kg), gentamicin (100 mg/kg) or azithromycin (30 mg/kg) once daily for ten consecutive days. Histopathological examination of tissues by transmission electron microscopy (TEM) indicated different degrees of accumulation of phospholipidosis in liver, lung, mesenteric lymph node, and kidney of drug-treated rats but not controls. Liquid chromatography coupled to mass spectrometry (LC/MS) was used to identify levels of endogenous biochemical profiles in rat urine. Urinary levels of di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) correlated with induction of phospholipidosis for amiodarone, gentamicin and azithromycin. Rats treated with gentamicin also had increased urinary levels of several phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) species.

Mitogenic action of lysophosphatidic acid in proximal tubular epithelial cells obtained from voided human urine.

Focal tubular cell multiplication at sites on an injured nephron is a critical event in the recovery phase following acute tubular necrosis. During this process, numerous viable tubular cells exfoliate and are shed into the urine. Lysophosphatidic acid (LPA) is generated in the plasma membrane of injured cells and acts as an intercellular mediator of various biological processes, including inflammation, proliferation and repair. In the present study, exfoliated proximal tubule (PT) cells were isolated from human urine and the mitogenic effects of LPA were investigated as a model of repair and proliferation following renal injury. LPA stimulated a 23. 5% increase in DNA synthesis, a 29.4% increase in cell number and an 86.6% decrease in cAMP content. All of these responses were pertussis toxin sensitive, indicating the involvement of G(i)-type G-proteins in LPA signalling. Conversely, the LPA-induced DNA synthesis and the decrease in intracellular cAMP content were insensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), suggesting a mitogenic response via PI3K-independent mechanisms. Furthermore, we detected specific mRNA transcripts for the recently cloned human LPA-receptors, endothelial differentiation gene (Edg)-2 and Edg-4 (Edg-2>>Edg-4) by reverse transcription-PCR in PT cells. Our data suggest that LPA may behave as a local growth factor in PT cells following tubular injury.

Abnormal excretion of urinary phospholipids and sulfatide in patients with mitochondrial encephalomyopathies.

We found that patients with mitochondrial encephalomyopathies excreted urinary phosphatidylethanolamine, cardiolipin, and phosphatidylserine most likely derived from mitochondria and sulfatide which is specific to myelin or the kidney. It is of interest that four patients with myoclonus epilepsy with ragged-red fibers and one patient with chronic progressive external ophthalmoplegia all showed qualitatively similar abnormal excretion of such urinary lipids. It is conceivable that the urinary acidic phospholipids reflect abnormalities in the mitochondrial phospholipids, which are very important for mitochondrial enzymatic activities.