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Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues that play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter (UT)-B, UT-A1/A3, or all UTs leads to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UT-A1–knockout mouse model. Phenotypically, daily urine output in UT-A1–knockout mice was nearly 3-fold that of WT mice and 82% of all-UT–knockout mice, and the UT-A1–knockout mice had significantly lower urine osmolality than WT mice. After 24-h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1–knockout mice were unable to increase urine-concentrating ability. Compared with all-UT–knockout mice, the UT-A1–knockout mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UT-A1 represents a promising diuretic target.

Changes to extracellular matrix (ECM) structures are linked to tumor cell proliferation and metastasis. We previously reported that naturally occurring peptides of collagen type I are elevated in urine of patients with colorectal liver metastasis (CRLM). In the present study, we took an MS-based proteomic approach to identify specific collagen types that are up-regulated in CRLM tissues compared with healthy, adjacent liver tissues from the same patients. We found that 19 of 22 collagen-α chains are significantly up-regulated (p < 0.05) in CRLM tissues compared with the healthy tissues. At least four collagen-α chains were absent or had low expression in healthy colon and adjacent tissues, but were highly abundant in both colorectal cancer (CRC) and CRLM tissues. This expression pattern was also observed for six noncollagen colon-specific proteins, two of which (CDH17 and PPP1R1B/DARP-32) had not previously been linked to CRLM. Furthermore, we observed CRLM-associated up-regulation of 16 proteins (of 20 associated proteins identified) known to be required for collagen synthesis, indicating increased collagen production in CRLM. Immunohistochemistry validated that collagen type XII is significantly up-regulated in CRLM. The results of this study indicate that most collagen isoforms are up-regulated in CRLM compared with healthy tissues, most likely as a result of an increased collagen production in the metastatic cells. Our findings provide further insight into morphological changes in the ECM in CRLM and help explain the finding of tumor metastasis–associated proteins and peptides in urine, suggesting their utility as metastasis biomarkers.

During Escherichia coli urinary tract infections, cells in the human urinary tract release the antimicrobial protein siderocalin (SCN; also known as lipocalin 2, neutrophil gelatinase-associated lipocalin/NGAL, or 24p3). SCN can interfere with E. coli iron acquisition by sequestering ferric iron complexes with enterobactin, the conserved E. coli siderophore. Here, we find that human urinary constituents can reverse this relationship, instead making enterobactin critical for overcoming SCN-mediated growth restriction. Urinary control of SCN activity exhibits wide ranging individual differences. We used these differences to identify elevated urinary pH and aryl metabolites as key biochemical host factors controlling urinary SCN activity. These aryl metabolites are well known products of intestinal microbial metabolism. Together, these results identify an innate antibacterial immune interaction that is critically dependent upon individualistic chemical features of human urine.

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin β1 subunit in these cells converts them from a "loose" epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a "tight" epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin β1 cytoplasmic tail and does not entail β1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the β1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin β1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.

Thrombomodulin (TM) is an integral membrane glycoprotein, which occurs as both a chondroitin sulfate (CS) proteoglycan (PG) form (β-TM) and a non-PG form without a CS chain (α-TM) and hence is a part-time PG. An α-TM preparation isolated from human urine contained the glycosaminoglycan linkage region tetrasaccharide GlcUAβ1-3Galβ1-3Galβ1-4xylose, and the nonreducing terminal GlcUA residue is 3-O-sulfated. Because the human natural killer-1 sulfotransferase (HNK-1ST) transfers a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue in the HNK-1 antigen precursor trisaccharide, GlcUAβ1-3Galβ1-4GlcNAc, the sulfotransferase activity toward the linkage region was investigated. In fact, the activity of HNK-1ST toward the linkage region was much higher than that toward the glucuronylneolactotetraosylceramide, the precursor of the HNK-1 epitope. HNK-1ST may be responsible for regulating the sorting of α- and β-TM. Furthermore, HNK-1ST also transferred a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue of a chondroitin chain. Intriguingly, the HNK-1 antibody recognized CS chains and the linkage region if they contained GlcUA(3-O-sulfate), suggesting that HNK-1ST not only synthesizes the HNK-1 epitope but may also be involved in the generation of part-time PGs.

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (

Myeloperoxidase-derived HOCl targets tissue- and lipoprotein-associated plasmalogens to generate α-chlorinated fatty aldehydes, including 2-chlorohexadecanal. Under physiological conditions, 2-chlorohexadecanal is oxidized to 2-chlorohexadecanoic acid (2-ClHA). This study demonstrates the catabolism of 2-ClHA by ω-oxidation and subsequent β-oxidation from the ω-end. Mass spectrometric analyses revealed that 2-ClHA is ω-oxidized in the presence of liver microsomes with initial ω-hydroxylation of 2-ClHA. Subsequent oxidation steps were examined in a human hepatocellular cell line (HepG2). Three different α-chlorinated dicarboxylic acids, 2-chlorohexadecane-(1,16)-dioic acid, 2-chlorotetradecane-(1,14)-dioic acid, and 2-chloroadipic acid (2-ClAdA), were identified. Levels of 2-chlorohexadecane-(1,16)-dioic acid, 2-chlorotetradecane-(1,14)-dioic acid, and 2-ClAdA produced by HepG2 cells were dependent on the concentration of 2-ClHA and the incubation time. Synthetic stable isotope-labeled 2-ClHA was used to demonstrate a precursor-product relationship between 2-ClHA and the α-chlorinated dicarboxylic acids. We also report the identification of endogenous 2-ClAdA in human and rat urine and elevations in stable isotope-labeled urinary 2-ClAdA in rats subjected to intraperitoneal administration of stable isotope-labeled 2-ClHA. Furthermore, urinary 2-ClAdA and plasma 2-ClHA levels are increased in LPS-treated rats. Taken together, these data show that 2-ClHA is ω-oxidized to generate α-chlorinated dicarboxylic acids, which include α-chloroadipic acid that is excreted in the urine.

The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.

In the prion diseases, a prolonged, asymptomatic incubation period precedes the onset of neurologic dysfunction. At present, a noninvasive test is not available for the presymptomatic diagnosis of prion disease, and thus the report of a test for prions using urine has been of great interest (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482). Using Western immunoblots with the anti-prion protein (PrP) 3F4 monoclonal antibody and an anti-mouse IgG secondary antibody, a protease-resistant PrP was reported in the urine of Syrian hamsters and humans with prion disease. Here we have demonstrated that this purportedly "protease-resistant PrP" band in the urine of diseased hamsters is detectable using the anti-mouse IgG secondary antibody in the absence of the 3F4 monoclonal antibody. Mass spectrometric analysis identified an immunoglobulin light chain in the band but found no PrP peptides. No similar band was found in the urine of uninfected hamsters or in brain homogenates from normal or prion-infected hamsters. Moreover, the band in the urine of infected hamsters was not detected using two chimeric human-mouse recombinant anti-PrP antibody fragments followed by an anti-human IgG secondary antibody. Our results indicate that the band detected under previously published conditions is due to the cross-reactivity of the anti-mouse IgG antibody with IgG light chains and possibly heavy chain fragments in urine, but not with PrP.

OREBP (osmotic response element-binding protein), also called TonEBP or NFAT5, is thought to induce the expression of genes that increase the accumulation of organic osmolytes to protect cells against a hypertonic environment. To investigate the consequences of lacking OREBP activity, transgenic (Tg) mice that overexpress OREBPdn (dominant negative form of OREBP) specifically in the epithelial cells of the renal collecting tubules were generated. These mice showed impairment in their urine concentrating mechanism, most likely due to reduced expression of the aquaporin AQP2 and the urea transporter UT-A1 and UT-A2 mRNAs. When deprived of water or after the administration of a vasopressin analogue, urine osmolality of the Tg mice was significantly increased but not to the same extent as that of the wild type mice. The expression of AQP2 and UT-A1, but not UT-A2 mRNAs, was increased to the same level as that of the wild type mice in the water deprivation state, indicating that the vasopressin regulatory mechanism was not affected by OREBPdn. These data indicate that in addition to vasopressin, OREBP is another essential regulator of the urine concentrating mechanism. Furthermore, the OREBPdn Tg mice developed progressive hydronephrosis soon after weaning, confirming the osmoprotective function of OREBP implicated by the in vitro experiments.

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U(osm)) was lower (1532 +/- 71 versus 2056 +/- 83 mosM/kg H(2)O, mean +/- S.E., p < 0.001). After 24 h of water deprivation, U(osm) (in mosM/kg H(2)O) was 2403 +/- 38 in UT-B null mice and 3438 +/- 98 in wild-type mice (p < 0.001). Plasma urea concentration (P(urea)) was 30% higher, and urine urea concentration (U(urea)) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U(urea)/P(urea) ratio (61 +/- 5 versus 124 +/- 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "urea-selective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.

Aquaporins (AQPs) are channel proteins that regulate the movement of water through the plasma membrane of secretory and absorptive cells in response to osmotic gradients. In the salivary gland, AQP5 is the major aquaporin expressed on the apical membrane of acinar cells. Previous studies have shown that the volume of saliva secreted by AQP5-deficient mice is decreased, indicating a role for AQP5 in saliva secretion; however, the mechanism by which AQP5 regulates water transport in salivary acinar cells remains to be determined. Here we show that the decreased salivary flow rate and increased tonicity of the saliva secreted by Aqp5(-)/- mice in response to pilocarpine stimulation are not caused by changes in whole body fluid homeostasis, indicated by similar blood gas and electrolyte concentrations in urine and blood in wild-type and AQP5-deficient mice. In contrast, the water permeability in parotid and sublingual acinar cells isolated from Aqp5(-)/- mice is decreased significantly. Water permeability decreased by 65% in parotid and 77% in sublingual acinar cells from Aqp5(-)/- mice in response to hypertonicity-induced cell shrinkage and hypotonicity-induced cell swelling. These data show that AQP5 is the major pathway for regulating the water permeability in acinar cells, a critical property of the plasma membrane which determines the flow rate and ionic composition of secreted saliva.

Aquaporin (AQP) water channel AQP3 has been proposed to be the major glycerol and non-AQP1 water transporter in erythrocytes. AQP1 and AQP3 are also expressed in the kidney where their deletion in mice produces distinct forms of nephrogenic diabetes insipidus. Here AQP1/AQP3 double knockout mice were generated and analyzed to investigate the functional role of AQP3 in erythrocytes and kidneys. 53 double knockout mice were born out of 756 pups from breeding double heterozygous mice. The double knockout mice had reduced survival and impaired growth compared with the single knockout mice. Erythrocyte water permeability was 7-fold reduced by AQP1 deletion but not further reduced in AQP1/AQP3 null mice. AQP3 deletion did not affect erythrocyte glycerol permeability or its inhibition by phloretin. Daily urine output in AQP1/AQP3 double knockout mice (15 ml) was 9-fold greater than in wild-type mice, and urine osmolality (194 mosm) was 8.4-fold reduced. The mice remained polyuric after DDAVP administration or water deprivation. The renal medulla in most AQP1/AQP3 null mice by age 4 weeks was atrophic and fluid-filled due to the severe polyuria and hydronephrosis. Our data provide direct evidence that AQP3 is not functionally important in erythrocyte water or glycerol permeability. The renal function studies indicate independent roles of AQP1 and AQP3 in countercurrent exchange and collecting duct osmotic equilibration, respectively.

F(2)-isoprostanes (iPs) are free radical-catalyzed isomers of prostaglandin F(2alpha). Circulating and urinary iPs have been used as indices of lipid peroxidation in vivo. Utilizing an (18)O-labeled homologous internal standard, we developed a gas chromatography/mass spectrometry assay for the 2,3-dinor-5,6-dihydro (dinor-dihydro) metabolite of iPF(2alpha)-III. Although urinary excretion of iPF(2alpha)-III reflects systemic lipid peroxidation, the metabolite is more abundant (median of 877 (range of 351-1831) versus 174 (range of 56-321) pg/mg of creatinine; p0.01) than the parent iP in urine and can be measured in plasma. Metabolite analysis may be preferable in plasma due to the abundance of arachidonic acid as a source of ex vivo lipid peroxidation. Also, iPF(2alpha)-III may be formed in blood samples in a cyclooxygenase-dependent manner by platelets ex vivo. By contrast, the metabolite is not formed by aggregated platelets (0.71 +/- 0.08 versus 0.65 +/- 0.09 pg/ml). Although the metabolite/parent ratio is altered in cirrhosis, urinary dinor-dihydro-iPF(2alpha)-III is elevated and increases further during reperfusion following orthoptic liver transplantation. In addition to its formation as an iPF(2) metabolite, analysis of gamma-linolenic acid autooxidation products and the compound present in freeze-thawed plasma suggests that gamma-linolenic acid may also be an important source of dinor-dihydro-iPF(2alpha)-III.

O-linked sugar chains with xylose as a reducing end linked to human urinary soluble thrombomodulin were studied. Sugar chains were liberated by hydrazinolysis followed by N-acetylation and tagged with 2-aminopyridine. Two fractions containing pyridylaminated Xyl as a reducing end were collected. Their structures were determined by partial acid hydrolysis, two-dimensional sugar mapping combined with exoglycosidase digestions, methylation analysis, mass spectrometry, and NMR as SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1+ ++-4Xyl. These sugar chains could bind to an HNK-1 monoclonal antibody. This is believed to be the first example of a proteoglycan linkage tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.

Water channel aquaporin-1 (AQP1) is strongly expressed in kidney in proximal tubule and descending limb of Henle epithelia and in vasa recta endothelia. The grossly normal phenotype in human subjects deficient in AQP1 (Colton null blood group) and in AQP4 knockout mice has suggested that aquaporins (other than the vasopressin-regulated water channel AQP2) may not be important in mammalian physiology. We have generated transgenic mice lacking detectable AQP1 by targeted gene disruption. In kidney proximal tubule membrane vesicles from knockout mice, osmotic water permeability was reduced 8-fold compared with vesicles from wild-type mice. Although the knockout mice were grossly normal in terms of survival, physical appearance, and organ morphology, they became severely dehydrated and lethargic after water deprivation for 36 h. Body weight decreased by 35 +/- 2%, serum osmolality increased to >500 mOsm, and urinary osmolality (657 +/- 59 mOsm) did not change from that before water deprivation. In contrast, wild-type and heterozygous mice remained active after water deprivation, body weight decreased by 20-22%, serum osmolality remained normal (310-330 mOsm), and urine osmolality rose to >2500 mOsm. Urine [Na+] in water-deprived knockout mice was

Endogenous Na(+)-pump specific inhibitors are present in the plasma, urine, and tissues of humans and animals. To date, the source of these inhibitors has not been rigorously defined. In the present study, large amounts of several Na(+)-pump specific inhibitors have been demonstrated to exist in the urine of rats raised on a regular chow diet and tap water. All of the inhibitor levels have been found to increase 1.5-8-fold by the surgical preparation of reduced renal mass (RRM) and one-kidney, one-clip (IK, IC) hypertension. These urinary inhibitors, however, except for the ouabain-like inhibitor which eluted from a high performance liquid chromatography C18 column at the same retention time as [3H]ouabain, disappeared within a week after switching the diet from regular diet (number 5001, PMI Feeds, Inc.) to pure synthetic diet (number 5755). The urinary level of the ouabain-like inhibitor decreased to only one-half of the level in the control rat raised on a regular diet. Two of these inhibitors were purified from both urine and diet by a combination of Amberlite XAD-2 adsorption chromatography, reverse phase low pressure liquid chromatography, and several high performance liquid chromatographies. Reverse phase high performance liquid chromatography, liquid secondary ion and gas-liquid mass spectrometries, and proton nuclear magnetic resonance spectroscopy identified these inhibitors as a stereoisomer of convalloside, probably neoconvalloside, and a mono-rhamnoside of periplogenin or its stereoisomer. These cardiac glycosides exhibited inhibitory potencies comparable to ouabain against ouabain-displacement from Na+,K(+)-ATPase and against 86Rb uptake into human erythrocytes, and they also exhibited cross-reactivity to anti-ouabain antibodies and anti-digoxin antibodies. These results clearly demonstrate that the principal source of most of the inhibitors in rat urine is the diet. The results suggest that the ouabain-like inhibitor may be derived from an endogenous origin.

2-Keto-3-methylvaleric acid (KMVA) has been found not to undergo spontaneous keto-enol tautomerization in neutral aqueous solution, alone or in the presence of large concentrations of pyridoxamine or pyridoxamine-5-phosphate. This finding denies the commonly held suppositions that 3R-KMVA is derived spontaneously from 3S-KMVA in vivo, and that L-alloisoleucine is the product of the reamination of this 3R-KMVA. Evidence presented here suggests that racemization of the 3-carbon of L-isoleucine occurs during transamination, that L-alloisoleucine is an inherently unavoidable by-product of L-isoleucine transamination (and vice versa), and that a KMVA enol is not obligate in this racemization. The four stereoisomers of 2-hydroxy-3-methylvaleric acid have been synthesized and the mass spectra of their trimethylsilyl derivatives recorded. An achiral methylsilicone column was used to separate the diastereomeric pairs and to determine their relative ratios in plasma and urine from normal controls and two maple syrup urine disease (MSUD) patients. The urinary ratio of the two diastereomers is different from that for plasma, both in normals and in MSUD patients. The plasma ratios may provide a rapid and simple measure of residual branched chain 2-keto acid dehydrogenase activity in MSUD patients.

The excretion rates of different oligosaccharides in the urine of two individuals, a blood group O, nonsecretor woman, and a blood group A, secretor woman, were studied at different times during pregnancy and lactation. Quantitative determinations of di-, tri-, and tetrasaccharides were performed by combined gas-liquid chromatography-mass spectrometry. Larger oligosaccharides were quantitated by their content of total hexose or 6-deoxyhexose (fucose). The urinary oligosaccharide excretion during pregnancy is detectable by the 13th week. All oligosaccharides studied increased and reached the highest levels during the last trimester. During lactation more complex excretion patterns were observed. Lactose and oligosaccharides with a lactose backbone were the most predominant compounds. Oligosaccharides with a lacto-K-tetraose or a lacto-N-neotetraose backbone were present both during pregnancy and lactation, but the former type showed a much more pronounced increase of excretion during lactation. Four oligosaccharides containing only D-glucose were found in largest amounts during pregnancy. One of these oligosaccharides, a glucose tetrasaccharide, was quantitatively determined in the urine of 13 individuals and the excretion was found to decrease 2- to 5-fold in the first week post partum relative to the last week of pregnancy. Urine from a woman whose fetus died unexpectedly in the 24th week of pregnancy was also studied. No oligosaccharides characteristic of a normal pregnancy could be detected, suggesting that at least some oligosaccharides appearing specifically during pregnancy might reflect the function of the placenta or the fetus, or both.

A corrected method for the measurement of pyrrole-2-carboxylate in rat urine was used in studies of its excretion under various experimental conditions. The findings implicated administered hydroxy-L-proline as a relatively efficient source of urinary pyrrole-2-carboxylate and tended to exclude administered L-proline as a significant direct source. Removal of aerobic gut flora had no influence on the excretion of pyrrole-2-carboxylate either endogenously or following hydroxy-L-proline administration. Related studied showed that rat kidney L-amino acid oxidase catalyzes oxidation of hydroxy-L-proline to delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate on acidification of reaction mixtures. All findings were consistent with hydroxy-L-proline as the source of endogenous pyrrole-2-carboxylate excretion. Excretion patterns and labeling patterns were compared after administration of pyrrole-2-carboxylate or of hydroxy-proline epimers. From these data, the true excretion product of hydroxy-L-proline oxidation by L-amino acid oxidase appeared to be the unstable oxidation product, delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate in urine. The capacity of homogenates of guinea pig kidney and human kidney to carry out oxidation of hydroxy-L-proline to pyrrole-2-carboxylate was much less than that of rat kidney, consistent with the lower levels of urinary pyrrole-2-carboxylate in these species. Experiments designed to examine the modest increase of pyrrole-2-carboxylate excretion after proline loads led to new observations on tissue levels of hydroxy-L-proline following proline administration and on the inhibition by L-proline of hydroxy-L-proline oxidase.

Carnitine biosynthesis was studied in man and rat. Three healthy adult men were given intravenous injections of 1 mCi of [methyl-3H]epsilon-N-trimethyl-L-lysine, a precursor of carnitine. Labeled metabolites of this compound were monitored in serum and urine at 2, 6, 12, 24, and 48 h. At least nine radioactive metabolites were detected. For each collecton period, the specific activity of urinary carnitine exceeded the average serum specific activity. In man, the amount of labeled carnitine in urine was 2 to 8 times greater than labeled gamma-butyrobetaine (the immediate precursor of carnitine). In similar experiments in rats (intravenous injection of 0.1 mCi of [methyl-3H]epsilon-N-trimethyl-L-lysine), the specific activity of carnitine in urine was always lower than the corresponding average specific activity in serum. Between 0 and 2 h after administration of labeled precursor, the animals excreted large amounts of labeled gamma-butyrobetaine but little labeled carnitine. Significant gamma-butyrobetaine, 2-oxoglutarate dioxygenase (EC 1.14.11.1) activity was found in human kidney but this activity was absent in rat kidney. The results indicate that in man and rat the kidney accumulates intravenously administered [methyl-3H]epsilon-N-trimethyl-L-lysine. This compound is metabolized predominantly to gamma-butyrobetaine in rat kidney and to carnitine in human kidney. In both species, the synthesized products are at least partially leaked (either by secretion or by passive diffusion down a concentration gradient) into the renal tubular lumen from which they are either reabsorbed into the circulation for distribution to other tissues or excreted.

Profiles of 9 alpha, 11 alpha, 15(S)-trihydroxyprosta-5,13-dienoic acid (PGF2 alpha) and its catabolites in blood and urine of male adult Wistar rats were determined by high pressure liquid chromatography during and after slow intravenous infusion of tritium-labeled PGF2 alpha. In addition to PGF2 alpha and 15-keto-13,14-dihydro-PGF2 alpha, a major catabolite in blood during the infusion was identified as tetranor-15-keto-13,14-dihyro-PGF2 alpha (VII). This product was absent in the corresponding urine samples. VII was still detected in blood 2 h after the infusion was terminated. VII was previously shown by us to be an intermediate in the formation of certain urinary products of PGF2 alpha. These other catabolites of PGF2 alpha were also observed in both blood and urine although their levels were greater in the urine. Our observations indicate that a long lasting catabolite of PGF2 alpha, i.e. VII, as well as all the urinary catabolites of PGF2 alpha appear in the circulation. These results suggest that these catabolites are mostly (although not exclusively) extrarenal in origin (probably all hepatic) entering the circulation prior to their excretion by the kidney.

Male C57BL/6J mice were shown to excrete in their urine approximately 50 times as much glycolipid as do female mice. These lipids were primarily galabiglycosylceramides and globotriglycosylceramides which contained nonhydroxy and hydroxy fatty acids, sphingosine, and phytosphingosine as components of their ceramide moieties. The lipids were shown to be associated with multilamellar bodies. Neutral glycosphingolipids from kidney and urine were separated and analyzed by high performance liquid chromatographic techniques. Male kidneys were shown to contain about twice as much neutral glycosphingolipid as female kidneys. The female kidneys were virtually devoid of galactosylceramides and galabiglycosylceramides. Implantation of testosterone pellets in female mice resulted in a concomitant increase in urinary glycosphingolipids (greater than 100-fold) and in urinary lysosomal enzymes (about 10-fold). In mutant light ear mice, which are defective in excretion of lysosomal enzymes, the urinary excretion of glycolipids is also blocked. Taken together these data suggest that testosterone induces the formation of multilamellar kidney lysosomes which contain a complement of glycosphingolipids and which are normally excreted in the urine.

On amino acid analysis of urine of histidinemic patients, an unidentified compound was eluted in a position between beta-aminoisobutyric acid and gamma-aminobutyric acid. This compound was purified to homogeneity from the urine by a combination of extraction with 80% ethanol, repeated column chromatography on Bio-Rad AG-50, and high performance liquid chromatography on a strongly cationic ion exchanger. The compound yielded free histidine on hydrolysis in an evacuated sealed tube with 0.1-6.0 M HCl at 145 degrees C for 5 h, but not at 100 degrees C for 24 h. This compound was determined to be N tau-ribosylhistidine by 1H and 13C NMR spectroscopies. The urinary content of this material in normal and histidinemic children was 17.8 +/- 13.4 (n = 10) and 126 +/- 51 (n = 14) mumol/g creatinine (mean +/- S.D.), respectively, and were closely correlated with those of urinary histidine. The renal clearance value of N tau-ribosylhistidine in humans was 96% of that of creatinine. When rats were fed on diets rich in histidine, the urinary excretion of N tau-ribosylhistidine increased greatly and was well correlated with the intake of histidine.

Nine oligosaccharide fractions were purified from the urine of mannosidosis patients by Bio-Gel P-4 column chromatography, paper chromatography, and paper electrophoresis. Studies using exoglycosidase digestion, methylation analysis, periodate oxidation, and acetolysis revealed that most of the fractions were mixtures of two or three isomeric oligosaccharides. The structures of a total of 16 oligosaccharides were elucidated to be as follows: (formula: see text).

50 microCi of [3H]prostaglandin D2 tracer (100 Ci/mmol) was infused intravenously into a normal human male volunteer. 75% of the infused radioactivity was excreted into the urine within 5 h. This urine was added to urine obtained from two mastocytosis patients with marked overproduction of prostaglandin D2. Radiolabeled prostaglandin D2 urinary metabolites were chromatographically isolated and purified and subsequently identified by gas chromatography-mass spectrometry. 25 metabolites were identified. 23 of these compounds comprising 37% of the recovered radioactivity had prostaglandin F-ring structures, and only two metabolites comprising 2.7% of the recovered radioactivity retained the prostaglandin D-ring structure. The single most abundant metabolite identified was 9,11-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid which was isolated in a tricyclic form as a result of formation of a lower side chain hemiketal followed by lactonization of the terminal carboxyl and the hemiketal hydroxyl. Different isomeric forms of several prostaglandin F-ring metabolites were identified. An isomer of prostaglandin F2 alpha was also excreted intact into the urine as a metabolite of prostaglandin D2. 15 PGF-ring compounds were treated with n-butylboronic acid and 13 failed to form a boronate derivative, suggesting that the orientation of the hydroxyl group at C-11 in these 13 metabolites is beta. This study documents that prostaglandin D2 is metabolized to prostaglandin F-ring metabolites in vivo in humans. These results also bring into question the accuracy of quantifying prostaglandin F2 alpha metabolites as a specific index of endogenous prostaglandin F2 alpha biosynthesis, as well as quantifying urinary prostaglandin F2 alpha as an accurate index of renal production of prostaglandin F2 alpha.

N tau-Methylimidazoleacetic acid, the histamine metabolite, and its isomer, N pi-methylimidazoleacetic acid, were demonstrated and measured in rat brain and in human cerebrospinal fluid, urine, and plasma by a gas chromatographic-mass spectrometric method that is simple and specific with a detection limit of about 7 pmol (i.e. 1 ng). The acids were separated in biological samples by ion exchange chromatography, derivatized as n-butyl esters with boron trifluoride-butanol, and extracted with chloroform. Complete chemical ionization mass spectra and mass ion abundance ratios established the identity of N tau - and N pi - methylimidazoleacetic acids in the biological extracts and of imidazoleacetic acid in urine, but not in cerebrospinal fluid, plasma, and brain. The methylimidazoleacetic acids as n-butyl esters were quantified by electron impact selected ion monitoring of m/e 95 esters at different retention times. 3-Pyridylacetic acid was used as an internal standard and monitored at m/e 93. The levels of N tau-methylimidazoleacetic acid and N pi-methylimidazoleacetic acid were, respectively (picomoles/g or picomoles/ml +/- S.E.), for brain, 373, 19 +/- 13.08 and 110.33 +/- 12.44; for cerebrospinal fluid, 22.77 +/- 2.15 and 80.76 +/- 18.92; and for plasma, 84.57 +/- 13.64 and 73.64 +/- 14.50. In urine, the respective levels were 20.75 +/- 1.30 and 73.02 +/- 38.22 nmol/mg of creatinine. The origin of N pi-methylimidazoleacetic acid is not certain.

A method has been developed in which the human urinary prostaglandin metabolites are converted into stable prostanoic and prostanediotic acid homologues. The urine is reduced with sodium borohydride and the organic compounds, isolated with an Amberlite XAD-2 column, are treated with fuming hydrogen iodide, followed by treatment with zinc in methanolic hydrogen chloride, to yield dimethyl tetranorprostanedioate as the major product of human prostaglandin metabolism. This compound is determined by gas chromatography, after a single purification by thin layer chromatography. The excretion of total tetranorprostanedioic acid derivatives in healthy subjects was found to be 0.21 plus and minus 0.08 mg per g of creatinine or 0.3 plus and minus 0.01 mg per 24 hours.

In rats 80 to 95% of 4'-deoxypyridoxine administered intraperitoneally, intravenously, intramuscularly, or subcutaneously was excreted in the urine within 7.5 hours. Orally administered deoxypyridoxine was also rapidly eliminated. Over one-half of the excreted material appeared as deoxypyridoxine-3-(hydrogen sulfate) and the remainder as unchanged deoxypyridoxine. Tissue concentrations of deoxypyridoxine 5'phosphate were comparable to those of pyridoxal 5'phosphate. In normal men about 50% of a single oral dose (3 to 7.5 mg/kg of body weight) appeared in the urine within 6 hours. 4'Deoxy-5-pyridoxic acid accounted for 50 to 100% of the excreted material. The remainder was unchanged deoxypyridoxine. No deoxypyridoxine-3-(hydrogen sulfate) was detected in human urine and no 4'-deoxy-5-pyridoxic acid was found in rat urine. Deoxypyridoxine 5'-phosphate was not detected in the urine of either species. The complexity of deoxypyridoxine metabolism indicated by these data suggests the use of caution in extrapolating data obtained with deoxypyridoxine to B6 metabolism in the absence of deoxypyridoxine, and particularly in extrapolating results from the rat to man. Synthesis for 4'-deoxypyridoxine-3-(ethyl carbonate), 4'-deoxypyridoxine 5'-acetate, 4'-deoxy-3-0-(2-sulfoethyl)-pyridoxine, and the metabolites are presented. These synthesis were facilitated by using ethylchloroformate conjugates and N-methylpiperazine hydrolysis to block and unblock the phenol group.

The types of kinins excreted in fresh urine of dogs, rats, and humans were compared. Urinary kinins were separated by reverse-phase (C18) high performance liquid chromatography and quantitated by radioimmunoassay using an antibody directed against the COOH-terminal region of the peptide. Kinins were found in the following proportions: 53 +/- 3% bradykinin, 23 +/- 4% Lys-bradykinin, and 13 +/- 7% des-Arg1-bradykinin in dog urine; 67 +/- 6% bradykinin, 6 +/- 3% Lys-bradykinin, and 10 +/- 3% des-Arg1-bradykinin in rat urine; and 12 +/- 4% bradykinin, 30 +/- 3% Lys-bradykinin, 2 +/- 1% des-Arg1-bradykinin, and 41 +/- 3% unknown kinin in human urine. The unknown kinin was purified from a pool of human urine. Amino acid sequencing revealed a structure similar to Lys-bradykinin except that proline in position 4 was replaced by alanine ([Ala3]Lys-bradykinin). Synthetic and endogenous [Ala3]Lys-bradykinins had similar high performance liquid chromotography elution volumes and both had vasodilator activity and contracted the rat uterus. Human urinary kallikrein incubated with semipurified human low molecular weight kininogen released 76% of the total kinins as Lys-bradykinin, 7% as bradykinin, and 17% as [Ala3]Lys-bradykinin. In contrast, rat urinary kallikrein released 86% bradykinin, 18% Lys-bradykinin, and negligible amounts of [Ala3]Lys-bradykinin. The study revealed the presence of a new kinin, [Ala3]Lys-bradykinin, in human urine and it also proves that the types of kinins generated intrarenally are species-dependent.

Oligosaccharide patterns obtained by gel filtration of the urine of GM1-gangliosidosis Type 1 patients are quite different from those of GM1-gangliosidosis Type 2. By studies of oligosaccharides in the four major peaks obtained from the Type 1 subgroup using sequential exoglycosidase digestion, methylation analysis, and periodate oxidation, the structures of 15 oligosaccharides: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc, Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6(Gal beta 1 leads to 4Glc NAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6, and 3(Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6)Man beta 1 leads to 4GlcNAc, (formula see text) were elucidated. The amounts of total oligosaccharides excreted in the urine of the Type 2 subgroup were approximately one-tenth of those of Type 1. Moreover, the last eight oligosaccharides shown above, which have a Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to outer chain, were completely missing in the urine of Type 2.

A sleep-promoting substance, Factor S, has been extracted and purified from large volumes of human urine. Urinary Factor S is a small glycopeptide; amino acid-amino sugar analyses of the purified material revealed a substance composed of glutamic acid, alanine, diaminopimelic acid, and muramic acid in molar ratios of 2:2:1:1. The active glycopeptide resembles bacterial peptidoglycans but the composition suggests that it is not simply of adventitious origin. Other reasons for this conclusion are also given. Infusions into the lateral ventricle of the brain of about 5 pmol/kg of body weight induce a 50% increase in slow wave sleep in rabbits. The excess sleep is normal as judged by electrophysiological and behavioral criteria; it resembles the deep sleep that occurs when animals are allowed to sleep following prolonged sleep deprivation.

We have developed a highly sensitive and specific solid-phase enzyme immunoassay for 9-deoxy-delta 9,delta 12-dihydroprostaglandin D2 (delta 12-PGJ2) and studied the occurrence of this novel PGD2 metabolite in human urine. The assay detected delta 12-PGJ2 over the range of 2-200 pg, and the antiserum showed 2% cross-reaction with PGJ2 and less than 0.2% with other PGs. We used this assay and purified the delta 12-PGJ2-like immunoreactive substance from human urine. Purification consisted of chromatographies on a Sep-Pak C18 cartridge, a silicic acid column, reversed-phase high-performance liquid chromatography, and finally an affinity column of anti-delta 12-PGJ2 antibody. As a result, about 850 ng of delta 12-PGJ2-like immunoreactive substance were recovered from 60 liters of human urine. The purified material was identified as delta 12-PGJ2 by gas chromatography/high resolution-selected ion monitoring using the molecular ion m/z 448[M]+. and ions [M - 15]+, [M - 43]+, [M - 100]+., and [M - 143]+. The amounts of delta 12-PGJ2 in the urine from normal, volunteer men and women were 151.5 +/- 20.0 and 65.6 +/- 5.4 ng/24 h (mean +/- S.E., n = 5), respectively. The delta 12-PGJ2 amount in urine did not alter significantly during storage for at least 24 h or by the addition of authentic PGD2 to urine samples, suggesting that the delta 12-PGJ2 we determined was not derived from the decomposition of PGD2 in the urine during storage or purification. Moreover, when a single dose of PGD2 (1 mg/kg) was injected intravenously into cynomolgus monkeys, the urinary level of delta 12-PGJ2 increased 20- to 180-fold over the normal levels, whereas the delta 12-PGJ2 level decreased by 40-50% of the normal levels, following the administration of indomethacin at a dose of 1 mg/kg. These results indicate that delta 12-PGJ2 is formed naturally in the body and excreted as a urinary PGD2 metabolite.