Toward Noninvasive Diagnosis of IgA Nephropathy: A Pilot Urinary Metabolomic and Proteomic Study.

IgA nephropathy is diagnosed by renal biopsy, an invasive procedure with a risk of significant complications. Noninvasive approaches are needed for possible diagnostic purposes and especially for monitoring disease activity or responses to treatment. In this pilot project, we assessed the utility of urine samples as source of biomarkers of IgA nephropathy. We used spot urine specimens from 19 healthy controls, 11 patients with IgA nephropathy, and 8 renal-disease controls collected on day of renal biopsy. Urine samples were analyzed using untargeted metabolomic and targeted proteomic analyses by several experimental techniques: liquid chromatography coupled with mass spectrometry, immunomagnetic isolation of target proteins coupled with quantitation by mass spectrometry, and protein arrays. No single individual biomarker completely differentiated the three groups. Therefore, we tested the utility of several markers combined in a panel. Discriminant analysis revealed that combination of seven markers, three metabolites (dodecanal, 8-hydroxyguanosine, and leukotriene C4), three proteins (α1-antitrypsin, IgA-uromodulin complex, and galactose-deficient IgA1), and heparan sulfate, differentiated patients with IgA nephropathy from patients with other renal diseases and healthy controls. Future studies are needed to validate these preliminary findings and to determine the power of these urinary markers for assessment of responses to therapy.

Leukotrienes B4, C4, D4 and E4 in the exhaled breath condensate (EBC), blood and urine in patients with pneumoconiosis.

Leukotrienes (LTs) are involved in the pathogenesis of lung fibrosis and were increased in exhaled breath condensate (EBC) of the patients with pneumoconiosis. However the possible influence of extra-pulmonary disorders on the EBC markers is not known. Therefore in parallel with EBC, LTs' levels in the plasma and urine were measured in patients with pneumoconiosis (45 × asbestos exposure, 37 × silica exposure) and in 27 controls. Individual LTs B4, C4, D4 and E4 were measured by liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). In EBC, LT D4 and LT E4 were increased in both groups of patients (p<0.001 and p<0.05), comparing with the controls. Both LT B4 and cysteinyl LTs were elevated in asbestos-exposed subjects (p<0.05). Asbestosis with more severe radiological signs (s1/s2-t3/u2) and lung functions impairment has shown higher cysteinyl LTs and LT C4 in the EBC (p<0.05) than mild asbestosis (s1/s0-s1/s1). In addition, in the subjects with asbestosis, cysteinyl LTs in EBC correlated with TLC (-0.313, p<0.05) and TLCO/Hb (-0.307, p<0.05), and LT C4 with TLC (-0.358, p<0.05). In pneumoconioses, EBC appears the most useful from the 3 fluids studied.

Conjugated linoleic acid's lack of attenuation of hyperpnea-induced bronchoconstriction in asthmatic individuals in the short term.

PURPOSE: Conjugated linoleic acid (CLA) has been reported to modify the inflammatory response associated with allergic airway disease, primarily in animal models. To extend these observations to humans, the effect of short-term CLA supplementation on the severity of exercise-induced bronchoconstriction (EIB) was investigated in asthmatics. METHODS: Six subjects with physician-diagnosed asthma and EIB began the study on their usual diet, to which was added 4.8 g CLA/d for 8 wk. Pulmonary-function tests were administered before and after eucapnic voluntary hyperventilation (EVH) challenge at the commencement (Week 0) and conclusion of the treatment period (Week 8). Pre- and 90 min post-EVH challenge, urine was assayed for the presence of cysteinyl leukotrienes (LT) C4-E4 and 9α, 11ß-prostaglandin (PG) F2. RESULTS: Pre- to post-EVH forced expiratory volume in 1 s (FEV1) did not significantly differ (p > .05) from Week 8 to Week 0. The pre- to post-EVH decline in FEV1 at Week 8 (-29.6% ± 6.6%) was not significantly different (p > .05) from that at Week 0 (-32.0% ± 5.5%). Area under the curve of FEV1 plotted against time from zero to 60 min (AUC0-60) was unaltered at Week 8 (-931% ± 350% change per minute) compared with Week 0 (-1,090% ± 270% change per minute). CLA supplementation did not alter forced midexpiratory flow, forced vital capacity (FVC), or FEV1/FVC. In addition, post-EVH urinary LTC4-E4 and 9α, 11ß-PGF2 were unchanged after CLA supplementation. CONCLUSION: Daily supplementation of 4.8 g CLA for 8 wk does not attenuate airway inflammation or hyperpnea-induced bronchoconstriction in asthmatic individuals.

A patient with neurological symptoms and abnormal leukotriene metabolism: a new defect in leukotriene biosynthesis.

A 15-year-old male patient presented with mental retardation, mild motor impairment, and partial deafness. Biochemical investigations showed an abnormal urinary profile of leukotrienes. Concentration of leukotriene D(4) (LTD(4)), which is usually not detectable, was highly increased, whereas LTE(4), the major urinary metabolite in humans, was completely absent. These data suggest membrane-bound dipeptidase deficiency, a new defect in leukotriene biosynthesis on the step of LTE(4) synthesis, as underlying defect.

Synthesis and metabolism of leukotrienes in gamma-glutamyl transpeptidase deficiency.

Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC(4), the primary cysteinyl LT, is cleaved by gamma-glutamyl transpeptidase (GGT), resulting in LTD(4). We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC(4) (12.8-17.9 nmol/mol creatinine; controls, <0.005 nmol/mol creatinine), whereas LTE(4) was below the detection limit (<0.005 nmol/mol creatinine; controls, 32.2 +/- 8.6 nmol/mol creatinine). In plasma of one patient, LTC(4) was found to be increased (17.3 ng/ml; controls, 9.6 +/- 0.4 ng/ml), whereas LTD(4) and LTE(4) were below the detection limit (<0.005 ng/ml). LTB(4) was found within normal ranges. In contrast to controls, the synthesis of LTD(4) and LTE(4) in stimulated monocytes was below the detection limit (<0.1 ng/10(6) cells; controls, 37.1 +/- 4.8 cells and 39.4 +/- 5.6 ng/10(6) cells, respectively). The formation of [(3)H]LTD(4) from [(3)H]LTC(4) in monocytes was completely deficient (<0.1%; controls, 85 +/- 7%). Our data demonstrate a complete deficiency of LTD(4) biosynthesis in patients with a genetic deficiency of GGT. GGT deficiency represents a new inborn error of cysteinyl LT synthesis and provides a unique model in which to study the pathobiological coherence of LT and glutathione metabolism.

Cysteinyl leukotrienes in the bile of patients with obstructive jaundice.

BACKGROUND: Cysteinyl leukotrienes (LTs) are potent proinflammatory mediators. They are predominantly excreted from blood by hepatobiliary elimination. To explore the clinical significance of biliary cysteinyl LTs, we determined their concentration changes in bile during treatment in patients with obstructive jaundice. METHODS: Bile samples were obtained during endoscopic or transhepatic biliary drainage. Leukotrienes C(4), D(4), and E(4) were quantified by two-step reversed-phase high-performance liquid chromatography and subsequent radioimmunoassay. RESULTS: The increased excretion of cysteinyl LTs (LTC(4) + LTD(4) + LTE(4)) decreased between day 1 and 14 after drainage (means, 171 pmol/h to 79 pmol/h; P < 0.02). During drainage, the excretion was higher when there was additional cholangitis (mean, 225 and 86 pmol/h, with and without cholangitis, respectively; P < 0.001). The concentrations of LTD(4) and LTE(4) were also higher with additional cholangitis than without (LTD(4), mean 6.0 vs 2.0 nM; P < 0.05; LTE(4), 6.8 vs 2.4 nM; P < 0.02, respectively). Biliary LTC(4) was detected only in patients with cholangitis. The biliary excretion of cysteinyl LTs was positively correlated with leukocyte concentration ( r = 0.68; P < 0.005) and C-reactive protein ( r = 0.73; P < 0.005) in blood. Furthermore, only in the absence of cholangitis, the excretion was positively correlated with serum gamma-glutamyl transferase ( r = 0.76; P < 0.02) and alanine aminotransferase ( r = 0.72; P < 0.02). CONCLUSIONS: The excretion of biliary cysteinyl LTs increases with the severity of cholestasis and hepatic inflammation in patients with obstructive jaundice. An additional increase of cysteinyl LTs was observed during bacterial cholangitis. The increased biliary excretion of biologically active cysteinyl LTs may contribute to the aggravation of cholestasis and inflammatory reaction in obstructive jaundice.

Bee venom induces high histamine or high leukotriene C4 release in skin of sensitized beekeepers.

Histamine is the principal mediator released in the skin during immediate bee venom allergy but the significance of cysteinyl leukotrienes in these reactions is not known. We measured skin histamine and cysteinyl leukotriene release induced by bee venom in six sensitized beekeepers with the skin microdialysis technique. The skin was dialyzed for 2 h after skin prick test with bee venom, and the release of histamine and leukotriene C4 (LTC4) into the microdialysis fractions was measured. Leukotriene E4 (LTE) and methylhistamine excretion into the urine was assayed and whole blood histamine release test was performed. The release of histamine in the skin was variable: either high delayed, high immediate and delayed, weak release or no marked release. The histamine releasability in the skin correlated with that in whole blood. The three subjects with low histamine release exhibited high LTC4 release in the skin as well as high LTE4 excretion into the urine. Thus, the histamine and LTC4 releases were inversely associated with each other. These differences may explain the variation in the clinical reaction by bee stings in sensitized beekeepers.

Persistent increase in plasma and urinary leukotrienes after acute asthma.

Leukotrienes may mediate bronchoconstriction in asthma. Cysteinyl leukotriene production rises in vivo after allergen challenge, but few reports describe leukotriene concentrations in clinical asthma or in children. Using high performance liquid chromatography/radioimmunoassay, plasma and urinary leukotrienes in asthmatic children (aged 5-10 years) were measured during an acute exacerbation (peak expiratory flow (PEF) < 65%, n = 10) and one month later (PEF 74-169%, n = 9), and in non-atopic normal children (aged 1.3-13.2 years). In the asthmatics, geometric mean (95% confidence interval) plasma leukotriene B4 (LTB4) was 746 pg/ml (398 to 1403) acutely and 1026 pg/ml (662 to 1593) in remission, compared with 369 pg/ml (167 to 728) in the normal children (n = 14). Plasma cysteinyl leukotrienes were low or undetectable, but urinary leukotriene E4 (LTE4) was higher in the asthmatics during an acute episode (210 pmol/mmol creatinine, 101 to 454) and at follow up (179 pmol/mmol, 110 to 293), compared with the normal children (98 pmol/mmol, 81 to 118, n = 41). This persistent increase in plasma LTB4 and urinary LTE4 concentrations one month after a severe asthmatic episode suggests leukotriene production is related to chronic inflammation rather than to acute bronchoconstriction.

The role of prostanoids in the complications of extracorporeal shock wave lithotripsy (ESWL) in children.

ESWL is a safe and effective first-line treatment for urinary tract stone disease (UTSD) in children. The major complications arising from this procedure were upper urinary tract obstruction and ureteral colic. It was shown that prostaglandin synthetase inhibitors were effective in the treatment of urethral colic. The aim of this study was to measure urinary and plasma prostaglandin E2 (PGE2)- and leukotriene C4 (LTC4)-like activity in the patients who underwent ESWL before and after the treatment and investigate the role of cyclooxygenase (CO) and lipoxygenase (LO) products in early and late complications of ESWL. Urinary PGE2-like activity were increased 1 h after ESWL. (1.19 +/- 0.12 vs 1.59 +/- 0.15 g/ml, p < 0.02). The plasma values were decreased significantly after the treatment (16.7 +/- 1.7 vs 11.6 +/- 1.2 g/ml, p < 0.005). Urinary and plasma LTC4-like activities were found to be significantly decreased in the post-ESWL samples (0.58 +/- 0.006 vs 0.39 +/- 0.04, p < 0.002; 8.6 +/- 0.9 vs 4.2 +/- 0.6, p < 0.001, respectively). In conclusion, ESWL may stimulate the release of PG from the urinary tract resulting in increased peristaltism and the passage of stone fragments into the bladder. As this group of drugs has also nephrotoxic effects, they can be given prophylactically only to selected patients.

Elevated cysteinyl leukotriene excretion in experimental glomerulonephritis.

The involvement of cysteinyl leukotrienes (LT) in the etiology of glomerulonephritis (GN) was investigated in a rat model of nephrotoxic serum nephritis in which renal function, morphology, LTC4 synthase activity and urinary cysteinyl LT excretion were monitored over seven days. Significant alterations in renal function and morphology were evident on day 1 in nephritic rats, with a 12% decline in creatinine clearance, a greater than three-fold increase in urinary protein excretion and histologic evidence of basement membrane thickening. Urinary LTC4 excretion in the nephritic rats was elevated at this time to 140 +/- 38 pg/hr (P < 0.01) compared to undetectable levels in control animals. On days 3 and 7, while proteinuria intensified and glomerular filtration remained depressed, LTC4 excretion declined 14% (NS) and 79% (P < 0.05), respectively. The temporal changes in urinary LTC4 excretion were paralleled by concomitant alterations in LTC4 synthase activity in renal cortical microsomes, where an 84% (P < 0.01) drop in enzyme activity occurred from day 1 to day 7 in the nephritic group. This data provides the first measurement of urinary cysteinyl LT excretion and altered LTC4 synthase activity in a model of experimental GN and supports an early role for LT's in the development of subsequent functional changes.

Plasma and urinary leukotrienes in sickle cell disease: possible role in the inflammatory process.

Sickle cell (HbSS) disease is associated with rheological and inflammatory stresses within the microcirculation. In order to determine the role of leukotrienes in the inflammatory processes in HbSS patients, we analysed plasma and urine levels of leukotrienes (LT); LTB4, LTC4, LTD4, and LTE4 as indicators of their in vivo metabolism. Plasma and urine level samples of 15 HbSS patients in steady-state and age-matched healthy, homozygous (HbAA) controls were extracted for leukotrienes and quantitated by HPLC. Control plasma level of leukotrienes (mean +/- SEM, ng ml-1) were: LTB4, 8.95 +/- 0.26; LTC4, 7.24 +/- 0.21; LTD4, 11.42 +/- 0.40; and LTE4, 14.51 +/- 0.50. Corresponding values for HbSS patients were: LTB4, 6.15 +/- 0.42; LTC4, 13.61 +/- 1.45; LTD4, 6.44 +/- 0.51 and LTE4, 4.97 +/- 0.37. The differences were significant at P < 0.05. Urine levels (mean +/- SEM, ng mmol-1 creatinine), for controls were: LTB4, 10.60 +/- 0.35; LTC4, 360.0 +/- 9.82. Values for HbSS urine were: LTB4, 27.50 +/- 3.33; LTC4, 356.0 +/- 17.87; LTD4, 69.90 +/- 14.51. LTD4 was not detected in control urine. These results suggest that sickle cell patients may exhibit impaired ability to catabolize LTC4 in plasma during steady state conditions. This altered metabolism may contribute to the persistent stress of the microcirculation, and is probably related to the abnormal microvascular rheology of sickle blood cells.