A Noninvasive Urine Metabolome Panel as Potential Biomarkers for Diagnosis of T Cell-Mediated Renal Transplant Rejection.

Acute T cell-mediated rejection (TCMR) is a major complication after renal transplantation. TCMR diagnosis is very challenging and currently depends on invasive renal biopsy and nonspecific markers such as serum creatinine. A noninvasive metabolomics panel could allow early diagnosis and improved accuracy and specificity. We report, in this study, on urine metabolome changes in renal transplant recipients diagnosed with TCMR, with a view to future metabolomics-based diagnostics in transplant medicine. We performed urine metabolomic analyses in three study groups: (1) 7 kidney transplant recipients with acute TCMR, (2) 15 kidney transplant recipients without rejection but with impaired kidney function, and (3) 6 kidney transplant recipients with stable renal function, using 1H-nuclear magnetic resonance. Multivariate modeling of metabolites suggested a diagnostic panel where the diagnostic accuracy of each metabolite was calculated by receiver operating characteristic curve analysis. The impaired metabolic pathways associated with TCMR were identified by pathway analysis. In all, a panel of nine differential metabolites encompassing nicotinamide adenine dinucleotide, 1-methylnicotinamide, cholesterol sulfate, gamma-aminobutyric acid (GABA), nicotinic acid, nicotinamide adenine dinucleotide phosphate, proline, spermidine, and alpha-hydroxyhippuric acid were identified as novel potential metabolite biomarkers of TCMR. Proline, spermidine, and GABA had the highest area under the curve (>0.7) and were overrepresented in the TCMR group. Nicotinate and nicotinamide metabolism was the most important pathway in TCMR. These findings call for clinical validation in larger study samples and suggest that urinary metabolomics warrants future consideration as a noninvasive research tool for TCMR diagnostic innovation.

Diagnostic performance evaluation of sulfate-conjugated cholesterol metabolites as urinary biomarkers of Niemann-Pick disease type C.

BACKGROUND: Niemann-Pick disease type C (NPC) is an autosomal recessive inherited disorder with progressive neuronal degeneration. Because conventional diagnostic methods are complicated and invasive, biomarker tests have drawn attention. We aimed to evaluate three urinary conjugated cholesterol metabolites as diagnostic biomarkers for NPC. METHODS: Urine samples from 23 patients with NPC, 28 healthy controls, and 7 patients with inherited metabolic disorders were analyzed. 3ß-Sulfooxy-7ß-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine conjugates in urine were quantified by liquid chromatography-tandem mass spectrometry. The diagnostic performance of the three metabolites and their total concentration was evaluated. RESULT: Creatinine-corrected concentrations of three metabolites and their total concentration were all significantly higher in NPC patients (0.0098 < P < .0448). The area under the receiver operating curve for all metabolites exceeded 0.95, the clinical specificity was 92-100%, and the clinical sensitivity was ~95%. In the urine of patients with other inherited metabolic diseases, the concentrations of the metabolites were lower than those in the urine of patients with NPC. CONCLUSION: These conjugated cholesterol metabolites in urine can serve as useful diagnostic markers for noninvasive screening of NPC.

Absolute quantification of cholesteryl esters using liquid chromatography-tandem mass spectrometry uncovers novel diagnostic potential of urinary sediment.

BACKGROUND: Urine has been utilized as a source of biomarkers in renal disease. However, urinary lipids have not attracted much attention so far. Here we studied urinary cholesteryl ester (CE) and its relevance in renal disease. METHODS: Quantitative analysis of CE molecular species in serum, urinary supernatant, and urinary sediment from patients with renal disease (N=64) and non-renal disease (N=23) was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and deuterated CEs as internal standards. RESULTS: Validation study showed good precision and accuracy of LC-MS/MS. Many CE species were detected in the urinary sediment and supernatant in the renal disease group, whereas only a few CE species were detected in the other group. In the renal disease group, the sum of the concentrations of all CE species showed a significant correlation between the sediment and the supernatant from urinary samples (r=0.876, p<0.001); however, the composition of CEs was significantly different between them. Further, the composition of CEs of the supernatant was similar to that of the serum. CONCLUSIONS: Our LC-MS/MS analysis uncovered a distinct CE profile in urinary sediment from patients with renal disease, suggesting a possible contribution of CEs in urothelial cells to the development of renal disease.

Physicochemical characterization of the urinary lipid from humans with nephrotic syndrome.

The aim of this study was to investigate the nature of urinary lipid in humans with nephrotic syndrome. Fresh urine specimens were fractionated by centrifugation into a lower cellular fraction and an upper noncellular fraction. Of the 11 urine specimens examined, six cellular fractions contained cells (oval fat bodies) that were laden with anisotropic (birefringent) droplets when viewed by polarizing microscopy. The mean total cholesterol excretions for the five urine specimens without anisotropic droplets and the six urine specimens with anisotropic droplets were 8.7 mg/L and 35.5 mg/L, respectively. The cellular fractions of the six urine specimens with anisotropic droplets are enriched in cholesterol esters relative to whole urine. Upon heating, the anisotropic droplets underwent phase transitions characteristic of cholesterol esters, as observed by polarizing microscopy. The mean cholesteric to isotropic phase transition temperature of the anisotropic droplets was 41.3 degrees C. These data indicate that the anisotropic droplets of the oval fat bodies were composed of cholesterol esters acylated largely with monounsaturated fatty acids consistent with cellular origin. The noncellular fractions were subjected to ultracentrifugation (48,000 X g for 2 hours) and then separated into supernate and infranate fractions. The supernate fractions contained minor amounts of lipid, except in two cases of massive lipiduria. In these two cases, the supernate fractions contained many individual anisotropic droplets with lipid composition nearly identical to their cellular fractions. The dispersed lipid of the infranate fractions were invisible by microscope. Nine of the 11 infranate fractions revealed an alpha migrating lipid band by agarose gel electrophoresis. For urine specimens with anisotropic droplets, the infranate fractions contained cholesterol ester fatty acids that were less saturated (as measured by gas-liquid chromatography) than the cellular fractions. Thus, the cholesterol esters of the infranate fractions were derived from a different source (probably high-density lipoproteins) than the cholesterol esters of the cellular fractions.

Excretion of (sulfated) steroids in the urine and excretion of cholesterol sulfate in the feces of boys with recessive X-linked ichthyosis.

The excretion of sulfated steroids was investigated in the urine and feces of six boys aged 9 months to 7 years and 10 months who had recessive X-linked ichthyosis. Profiles of urinary total steroids as well as sulfated steroids were normal. Cholesterol sulfate excretion in the urine was not elevated. In the feces 2-20% of total cholesterol was cholesterol sulfate, whereas in the feces of 28 healthy children no cholesterol sulfate was demonstrable. In the 6 patients total cholesterol excretion (500-2,500 mumol/kg feces) was also elevated in comparison with the 28 healthy controls (150-700 mumol/kg feces, mean 365 mumol/kg feces).

Studies on the clinical significance of nonesterified and total cholesterol in urine.

Gas-liquid chromatographic determinations of nonesterified and total urinary cholesterol were performed in 137 normals, 264 patients with various internal diseases without evidence of neoplasias or diseases of the kidney or urinary tract, 497 patients with malignancies and 236 patients with diseases of the kidney, urinary tract infections or prostatic adenoma with residual urine. A normal range (mean +/- 2 SD) of 0.2-2.2 mg/24 hours nonesterified cholesterol (NEC) and of 0.3-3.0 mg/24 hours total cholesterol (TC) was calculated. Values of urinary cholesterol excretion were independent of age and sex and did not correlate with cholesterol levels in plasma. Patients with various internal diseases, without evidence of neoplasias nor diseases of the kidney or obstruction of the urinary tract, showed normal urinary cholesterol excretions, as did patients with infections of the urinary tract. However, elevated urinary cholesterol was found in patients with diseases of the kidney or urinary tract obstruction (prostatic adenoma with residual urine), malignant diseases of the urogenital tract and metastasizing carcinoma of the breast. In patients with other malignant diseases urinary cholesterol was usually normal. Lesions of the urothelial cell membranes are considered to be the most likely cause of urinary cholesterol hyperexcretion. The clinical value of urinary cholesterol determinations as a possible screening test for urogenital carcinomas in unselected populations is limited by lacking specificity, expensive methodology and low prevalence of the mentioned carcinomas, although elevated urinary cholesterol excretions have been observed in early clinical stages of urogenital cancers.

Studies on the source of urinary cholesterol in the normal human male.

The aim of the present study was to obtain information on the source of urinary cholesterol in normal men of various age groups (22-25, 37-42, greater than 63 yrs). Aliquots of 24-hr urine samples were fractionated by ultracentrifugation. Between 10-20% of the total cholesterol in urine (measured by gas-liquid chromatography) separately sedimented after 2 x 10(4) g-min and after 4 x 10(5) g-min of centrifugation. However, an average of more than 50% of the total sedimented after 6 x 10(6) g-min and only 10-20% of total cholesterol remained in this supernatant. These results indicate that most of the cholesterol in urine of normal males is a component of a light particulate fraction. Little difference was seen in the distribution of urinary cholesterol among the various age groups examined The 4 x 10(5) g-min supernatant from 24-hour total urine samples was recentrifuged at 10(5) g for 60 min. The resulting pellet (100 P) was assayed for protein, total cholesterol and phospholipid. The cholesterol was 12-14% esterified. A molar ratio of total cholesterol to phospholipid of 1 to 0.8 was calculated. Assay of the 100 P fraction for marker enzymes showed an activity pattern characteristic for plasma membranes. Fractionation of the 100 P proteins by electrophoresis and separation of the 100 P phospholipids by thin-layer chromatography revealed patterns clearly different from those of human red cell plasma membrane. Electron micrographs of the 100 P fraction revealed an appearance similar to that of deteriorated membranes. The results suggest that most of the cholesterol in urine of the adult human male is present as a component of membranes derived from endogenous cells of the urogenital system.