Dynamic changes of urinary proteins in a rat model of acute hypercoagulable state induced by tranexamic acid.

The hypercoagulable state leads to the development of thrombotic diseases, but it is difficult to diagnose due to the lack of available biomarkers. This study aimed to investigate systematic changes of the urinary proteome in the acute hypercoagulable state. A rat model of the acute hypercoagulable state was induced by an antifibrinolytic agent tranexamic acid and urine samples were collected for proteomic analysis by liquid chromatography-tandem mass spectrometry. A total of 28 differential proteins were detected in the urinary proteome of the model rats, of which 12 had been previously considered as candidate biomarkers such as myoglobin, and 10 had been considered stable in healthy human urine. Of the 28 differentially expressed proteins 18 had counterparts in humans. Of these 18 proteins, 10 were members of the human core urinary proteome distributed in a variety of human tissues but concentrated in the urinary and digestive systems. Fumarylacetoacetase was verified as a potential marker of the acute hypercoagulable state by Western blot analysis. In conclusion, urine proteome analysis is a powerful approach to identify potential biomarkers of acute hypercoagulable state.

Activity-Based Protein Profiling of Intraoperative Serine Hydrolase Activities during Cardiac Surgery.

The processes involved in the initiation of acute kidney injury (AKI) following cardiopulmonary bypass (CPB) are thought to occur during the intraoperative period. Such a rapid development might indicate that some of the inductive events are not dependent on de novo protein synthesis, raising the possibility that changes in activities of pre-existing enzymes could contribute to the development of AKI. Activity-based protein profiling (ABPP) was used to compare the serine hydrolase enzyme activities present in the urines of CPB patients who subsequently developed AKI versus those who did not (non-AKI) during the intra- and immediate postoperative periods. Sequential urines collected from a nested case-control cohort of AKI and non-AKI patients were reacted with a serine hydrolase activity probe, fluorophosphonate-TAMRA, and separated by SDS-PAGE. The patterns and levels of probe-labeled proteins in the two groups were initially comparable. However, within 1 h of CPB there were significant pattern changes in the AKI group. Affinity purification and mass spectrometry-based analysis of probe-labeled enzymes in AKI urines at 1 h CPB and arrival to the intensive care unit (ICU) identified 28 enzymes. Quantitative analysis of the activity of one of the identified enzymes, kallikrein-1, revealed some trends suggesting differences in the levels and temporal patterns of enzyme activity between a subset of patients who developed AKI and those who did not. A comparative analysis of affinity-purified probe reacted urinary proteins from these patient groups during the intraoperative period suggested the presence of both shared and unique enzyme patterns. These results indicate that there are intraoperative changes in the levels and types of serine hydrolase activities in patients who subsequently develop AKI. However, the role of these activity differences in the development of AKI remains to be determined.

Sub-nephrotoxic cisplatin sensitizes rats to acute renal failure and increases urinary excretion of fumarylacetoacetase.

Nephrotoxicity limits the therapeutic efficacy of the antineoplastic drug cisplatin. Due to dosage adjustment and appropriate monitoring, most therapeutic courses with cisplatin produce no or minimal kidney damage. However, we studied whether even sub-nephrotoxic dosage of cisplatin poses a potential risk for the kidneys by predisposing to acute kidney injury (AKI), specifically by lowering the toxicity threshold for a second nephrotoxin. With this purpose rats were treated with a single sub-nephrotoxic dosage of cisplatin (3mg/kg, i.p.) and after two days, with a sub-nephrotoxic regime of gentamicin (50mg/kg/day, during 6 days, i.p.). Control groups received only one of the drugs or the vehicle. Renal function and renal histology were monitored throughout the experiment. Cisplatin treatment did not cause any relevant functional or histological alterations in the kidneys. Rats treated with cisplatin and gentamicin, but not those under single treatments, developed an overt renal failure characterized by both renal dysfunction and massive tubular necrosis. In addition, the urinary excretion of fumarylacetoacetase was increased in cisplatin-treated animals at subtoxic doses, which might be exploited as a cisplatin-induced predisposition marker. In fact, the urinary level of fumarylacetoacetase prior to the second nephrotoxin correlated with the level of AKI triggered by gentamicin in predisposed animals.

Evaluation of urinary biomarkers for the prognosis of drug-associated chronic tubulointerstitial nephritis.

BACKGROUND: The efficacy of urinary biomarkers for predicting adverse clinical outcomes in drug-induced chronic tubulointerstitial nephritis (D-CTIN) has not been well described. METHODS: A total of 54 patients with D-CTIN were prospectively followed-up in this study. The urinary excretion of α1-microglobulin and transforming growth factor-ß1 and the activity of urinary N-acetyl-ß-D-glucosaminidase (NAG) and matrix metalloproteinases (MMPs) 2 and 9 at baseline were measured. Changes in the estimated glomerular filtration rate (GFR) over a period of 11 to 54 months (median, 38 months) of follow-up were recorded. The efficacy of urinary biomarkers for differentiating patients with various outcomes was tested. Ten patients with IgA nephropathy and 20 healthy volunteers were enrolled as controls. RESULTS: The areas under the receiver operating characteristic curve for urinary NAG, MMP-9, MMP-2 and α1-microglobulin for predicting deterioration of the estimated GFR were 0.879, 0.867, 0.735 and 0.709, respectively (P < 0.05 for all). Partial regression coefficient results demonstrated that urinary NAG (P = 0.02), MMP-2 (P = 0.046) and MMP-9 (P = 0.041) were inversely correlated with the rate of GFR decline. CONCLUSIONS: Urinary NAG, MMP-2 and MMP-9 may be considered as possible candidates for forecasting the progression rate of D-CTIN.

Clinical, biochemical, and genetic analysis of a Korean neonate with hereditary tyrosinemia type 1.

BACKGROUND: Hereditary tyrosinemia type 1 (HT1; MIM 276700) is caused by mutations in the fumarylaceto-acetate hydrolase (FAH) gene, and is the most severe disorder associated with the tyrosine catabolic pathway. HT1 is a very rare disorder and no genetically confirmed case of HT1 in Korea has yet been reported. In this study, we present a Korean neonate with clinical and biochemical features of HT1. METHODS: A female neonate was admitted to our hospital for further work-up of an abnormal newborn screening test. We analyzed amino acids and organic acids in the patient's blood and urine. To confirm the presence of the genetic abnormality, all the coding exons of the FAH gene and the flanking introns were amplified by polymerase chain reaction (PCR). RESULTS: The patient's newborn screening test revealed increased concentrations of methionine and tyrosine. Subsequent urine organic acid analysis showed increased urinary excretion of 4-hydroxyphenyllactate, 4-hydroxyphenylpyruvate, succinate, and succinylacetone. Gap-PCR and sequence analysis of the FAH gene revealed a homozygous large deletion mutation encompassing exons 12-14. The patient's parents were not consanguineous but were heterozygous carriers of the same mutation. CONCLUSIONS: The patient had a novel, large deletion mutation of FAH and is the first report of genetically confirmed HT1 in Korea.

Chromatographic pattern classification.

In this paper, we propose and evaluate methodologies for the classification of images from thin-layer chromatography. Each individual sample is characterized by an intensity profile that is further represented into a feature space. The first steps of this process aim at obtaining a robust estimate of the intensity profile by filtering noise, reducing the influence of background changes, and by fitting a mixture of Gaussians. The resulting profiles are represented by a set of appropriate features trying to characterize the state of nature, here spread out over four classes, one for normal subjects and the other three corresponding to lysosomal diseases, which are disorders responsible for severe nerve degeneration. For classification purposes, a novel solution based on a hierarchical structure is proposed. The main conclusion of this paper is that an automatically generated decision tree presents better results than more conventional solutions, being able to deal with the natural imbalance of the data that, as consequence of the rarity of lysosomal disorders, has very few representative cases in the disease classes when compared with the normal population.

Simple and sensitive binding assay for measurement of adenosine using reduced S-adenosylhomocysteine hydrolase.

BACKGROUND: Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [(3)H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form. METHODS: SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid). RESULTS: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 +/- 2 nmol/L. Displacement of adenosine binding by the adenine 5'-nucleotides, adenine and hypoxanthine, required >1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC. CONCLUSIONS: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.

Expression of mouse uterine peptidylarginine deiminase in Escherichia coli: construction of expression plasmid and properties of the recombinant enzyme.

To study the structure/function relationships of peptidylarginine deiminase (PAD), we constructed an Escherichia coli expression plasmid for mouse uterine PAD. First, segments of a cDNA encoding murine PAD were subcloned into a single plasmid, and the resulting plasmid, pKSPAD1, was inserted into an expression vector, pKK223-3, at the EcoRI and HindIII restriction sites. Since no detectable amount or activity of the PAD was produced by E. coli carrying that plasmid, the 5'-untranslated sequence of the cDNA was replaced with several synthetic DNAs. One of the constructed plasmids, pKKPAD4, which had a unique DNA linker containing a pair of Shine-Dalgarno sequences and a short preceding cistron inserted into the adjacent 5'-region of the coding region, produced a large quantity of mouse PAD as an unfused protein in E. coli. The purified recombinant PAD was indistinguishable from the native enzyme with respect to some structural properties, such as molecular mass, amino- and carboxyl-terminal sequences, and circular dichroism spectra. However, the alpha-amino group of the amino-terminal methionine residue of the recombinant PAD was not acetylated as was that of the native enzyme. Comparison of the recombinant PAD with the natural enzyme did not indicate significant differences in their sensitivity to activation by Ca2+ and in their substrate specificity toward arginine derivatives. The rates of modification of soybean trypsin inhibitor (Kunitz) were also similar for the recombinant and native PADs. These results indicate that the recombinant PAD has biological activities identical to those of the native enzyme and that the N alpha-acetyl group in the native PAD does not appear to have any particular role in the enzyme's catalytic function.

Altered urinary excretion of lysosomal hydrolases in pregnancy.

Creatinine concentrations and the activities of five lysosomal hydrolases were measured in the serum and urine of 14 healthy nonpregnant control women and 19 healthy pregnant women. Fractional enzyme excretion (FEE) values for beta-glucuronidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, and alpha-mannosidase were calculated and compared between the two groups of subjects. Fractional enzyme excretion was calculated as the ratio of enzyme clearance to creatinine clearance. The FEE values for beta-galactosidase and alpha-mannosidase between the nonpregnant and pregnant populations were not statistically different; however, relative to the nonpregnant control group, the median FEE values for beta-glucuronidase (P < 0.03), beta-hexosaminidase (P < 0.06), and alpha-galactosidase (P < 0.02) were decreased approximately 1.5-, 1.8-, and 2.7-fold, respectively, in the pregnant population. The median urinary beta-galactosidase activity for the pregnant population, when expressed on the basis of creatinine, was twofold higher than that of the control group (P < 0.0005). These data indicate that with pregnancy there are marked changes in the urinary excretion of selected lysosomal enzymes, particularly alpha-galactosidase and beta-glucuronidase. When the molecular weights of these five hydrolases were compared between kidney homogenate and control urine, a correlation of 0.96 was observed, while the correlation between control serum and control urine was 0.69. This suggests that the FEE value differences between the pregnant and control groups are most likely due to changes in tubule cell metabolism, either decreased secretion or increased reabsorption. These biochemical changes may provide a means of assessing changes in renal function during pregnancy.

[The importance of the plasma proteins of the blood in maintaining the relative constancy of its hydrolytic properties]. / Znachenie belkov plazmy krovi v obespechenii otnositel'nogo postoianstva ee gidroliticheskikh svoistv.

Effects of hypervolemia, dehydration, activation and inhibition of urine-formation, i. v. administration of amylase and pepsinogen, experimental acute pancreatitis upon amylolytic activity of blood plasma, contents of pepsinogen in it, amylase and pepsinogen within the blood plasma protein factions, and excretion of enzymes with the urine, were studied in dogs. The data obtained suggest an important role of interconnection between amylase and pepsinogen with the plasma proteins in their renal and extrarenal excretion from the organism and in maintenance of a relative constancy of the contents and activity of enzymes in the blood. The affinity of the plasma proteins to their stains can indirectly characterise the transport capacity of the proteins in respect to amylase and pepsinogen.

Comparison of urinary excretion of four lysosomal hydrolases in healthy elderly and young adults.

The activities of four lysosomal enzymes and creatinine levels were measured in the plasma and urine of 17 healthy elderly and 7 young adults. Fractional enzyme excretion (FE ENZ) values for beta-hexosaminidase (N-acetylglucosaminidase), alpha-galactosidase, beta-galactosidase and beta-glucuronidase were calculated and compared between the two groups of subjects. FE ENZ was calculated as the ratio of enzyme clearance to creatinine clearance. The FE ENZ values for alpha-galactosidase, beta-galactosidase and beta-glucuronidase between the elderly and young populations were not statistically different; however, relative to the young control group, the FE ENZ value for beta-hexosaminidase was elevated approximately 2-fold in the elderly population (P = 0.06). The mean urinary alpha-galactosidase activity for the elderly population, when expressed on the basis of creatinine, was 50% lower than that of the control group (P = 0.03), whereas the mean urinary beta-hexosaminidase activity for the elderly was significantly higher compared to the control group (P = 0.008). When data for all subjects was analyzed, no correlation was observed between the urinary excretion of beta-hexosaminidase or alpha-galactosidase and glomerular filtration rate. These data indicate that with advancing age there are changes in the tubular secretion or reabsorption of selective lysosomal enzymes, particularly beta-hexosaminidase and alpha-galactosidase. These biochemical changes may provide a means of assessing subtle progressive deterioration of renal function.

The identification of type 1 Gaucher disease patients, asymptomatic cases and carriers in The Netherlands using urine samples: an evaluation.

The feasibility of using urine samples for the identification of patients with Gaucher disease and carriers has been investigated. It was found that the pH of a urine sample should be pH 6.0 or lower to ensure stability of lysosomal hydrolases. Two parameters of glucocerebrosidase, which is deficient in Gaucher disease, were studied using urine samples from control subjects, obligate carriers and patients. Firstly, the relative level of glucocerebrosidase activity was measured by relating the activity of the enzyme to that of another lysosomal hydrolase. Secondly, the enzymic activity of glucocerebrosidase per unit of protein was measured using an immunological method. The first method allowed discrimination of nearly all obligate carriers of type 1 Gaucher disease from normal individuals. The second method allowed clear discrimination of the majority of carriers from normal individuals, but some obligate carriers were not distinguishable from normal subjects on the basis of this parameter. However, the combination of both methods allowed discrimination between all obligate carriers examined so far (n = 34) and controls (n = 86). There was variability between healthy individuals in the relative amount of glucocerebrosidase in urine samples. A small proportion of healthy individuals have a relatively high activity of glucocerebrosidase in urine samples, reminiscent of observations made in white blood cells by other investigators. In urine samples from two unrelated parents of Gaucher disease patients a level of glucocerebrosidase activity was present that could not be distinguished from that in samples of patients. These individuals represent cases with subclinical manifestation of Gaucher disease, illustrating once more the remarkable heterogeneity in clinical expression of this disorder.

Excretion of urinary enzymes in female Sprague-Dawley rats in relation to cellular compartment, creatinine excretion and diuresis.

One hundred and one young-adult female Sprague-Dawley rats were acclimatized to metabolic cages for 2 days. After that time 24-hour urine was collected at a constant cooling temperature of 0-4 degrees C. After gel filtration the enzyme activities were determined, and the resulting values were used to calculate 24-hour excretions. The following reference ranges (2.5 and 97.5 percentiles) were determined (in mU/24 h): lactate dehydrogenase 43-181; phosphohexoseisomerase 45-1445; glutathione-S-transferase 1-299; alkaline phosphatase 27-1239; leucine arylamidase 72-377; gamma-glutamyltransferase 1334-9188; arylsulphatase A 59-309; beta-galactosidase 76-305; beta-glucuronidase 20-2756; beta-N-acetyl-D-glucosaminidase 66-491; glutamate dehydrogenase 7-711. There was a significant (though not very high) correlation with diuresis for the lysosomal enzymes beta-N-acetyl-D-glucosaminidase, arylsulphatase A and beta-galactosidase, and for glutamate dehydrogenase, lactate dehydrogenase, phosphohexoseisomerase and alkaline phosphatase. The relation to creatinine excretion was markedly close for the lysosomal enzymes beta-N-acetyl-D-glucosaminidase, arylsulphatase A and beta-galactosidase (r = 0.71-0.83), as well as for alkaline phosphatase, leucine arylamidase and gamma-glutamyltransferase. There was a relatively high correlation between the excretion of beta-N-acetyl-D-glucosaminidase, arylsulphatase A and beta-galactosidase among themselves (r = 0.63-0.81) as well as between leucine arylamidase and gamma-glutamyltransferase (r = 0.75).

Enzyme activities in urine: how should we express their excretion? A critical literature review.

Different methods and reference bases for characterizing the excretion of urinary enzymes (relation to volume, time, creatinine, specific gravity, osmolality and fractional excretion) are reviewed with regard to their advantages and disadvantages when using urinary enzyme excretion as a diagnostic tool. The problems of timed and untimed urine samples for the analysis of urinary enzyme excretion and the influence of diuresis on the quantity and composition of enzymes excreted are discussed. The use of the second morning urine as a random urine sample, and determination of the relation of enzyme activity to urinary creatinine (enzyme/creatinine ratio; enzyme/creatinine index) represent a satisfactory compromise for the characterization of urinary enzyme excretion. The calculation of enzyme excretion per unit time or other modes of expression should be used only in special cases.

Quality control material for activity determinations of urinary enzymes.

We examined the stability of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), and lysozyme (EC 3.2.1.17) in urine prepared by gel filtration and supplemented with albumin, or ethylene glycol, or ethylene glycol plus albumin during storage at -20 degrees C for a period of 12 months. The stability was assessed by linear regression analysis of monthly values versus time. All enzymes except for gamma-glutamyltransferase could be considered stable for about one year in all three control materials provided that maximum change of 10% of the starting enzyme activity is accepted as tolerable. If ethylene glycol is used as stabilizer, its suitability must be tested and its inhibitory effect on enzyme activities must be taken into account in intermethod comparisons, because in some methods, it may be removed in a pretreatment step.

Alterations of the tryptophan--nicotinic acid metabolism in vitiligo.

Tryptophan metabolism "via kynurenine" is altered in vitiligo: after a load of the amino acid the urinary excretion of 3-hydroxykynurenine and 3-hydroxyanthranilic acid is decreased, whereas that of xanthurenic acid and its 8-methyl ether is increased. The excretory values of the metabolites suggest a deficiency of the activity of kynurenine hydroxylase and kynunerinase, the enzymes involved in the metabolism of 3-hydroxykynurenine and 3-hydroxyanthranilic acid. The reduced excretion of 3-hydroxykynurenine, a tryptophan metabolite involved in melanin biosynthesis, may indicate a smaller utilization of tryptophan in the biogenesis of the melanins.

Urinary lysosomal hydrolases in mucolipidosis II and mucolipidosis III.

Investigation of the binding characteristics of acid beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase and alpha-L-fucosidase from patients with mucolipidosis II and mucolipidosis III to concanavalin A--Sepharose 4B revealed a 2--10-fold decrease in the proportion of enzyme activities from patients with mucolipidoses II and III that adsorbed on the lectin. Neuraminidase treatment of the unadsorbed enzyme fraction did not significantly increased the proportion of enzyme activities that bound to the concanavalin A--Sepharose 4B. Characterization of acid beta-D-galactosidase from the adsorbed and unadsorbed enzyme fractions of mucolipidosis II and mucolipidosis III patients demonstrated identical apparent Km values of 0.22 mM with respect to 4-methylumbelliferyl beta-D-galactopyranoside, altered pH--activity profiles and heterogeneous isoelectric-focusing patterns. The results of this study support the suggestion of an alteration of a post-translational modification (possibly glycosylation) occurring in mucolipidosis II and mucolipidosis III common to the lysosomal hydrolases that affects the mannoserelated properties of these enzymes.