Isoenzymes of four acid hydrolases in human kidney and urine

The occurrence of different isoenzymes of β-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, and α-mannosidase in human urine and kidney tissue was studied by isoelectric focusing. Artificial substrates were used for the enzymatic assays. There was a predominance of isoenzymes with a low isoelectric point in the urine. In the kidney tissue isoenzymes with higher isoelectric point predominated. This difference may be due to a higher proportion of N-acetylneuraminic acid-containing enzymes in the urine than in the kidney tissue.     

Calendar of forthcoming events

A simple and specific method for the determination of hydroxyproline in urine hydrolysates has been described. Hydroxyproline was converted into its N-isobutyloxycarbonyl methyl ester derivative without elaborate cleanup, which was analyzed by gas chromatography. Hydroxyproline was clearly separated from other urinary constituents on a 0.60% FFAP on dimethyldichlorosilane-treated Gas-Chrom P column. Kainic acid was used as the most convenient internal standard available. The relative standard deviations of peak height ratios were 1.15–2.51% at the 10–150;.μ levels. Percent recoveries of hydroxyproline added to urine hydrolysates ranged from 98.8 to 107.3%.     

β-p-Hydroxyphenylhydracrylic acid as a urinary constituent in a patient with gastrointestinal disease

In this paper the isolation of β-p-liydroxyphenylhydracrylic acid from urine of a patient with a severely impaired intestinal amino acid resorption is described. The compound has been identified by mass spectrometry. Mass spectrometry and gas chromatography parameters are given. The behaviour of the compound on twodimensional thin-layer (or paper) chromatograms is illustrated. β-p-hydroxyphenylhydracrylic aciduria must be very rare, even in patients with a severely impaired intestinal amino acid resorption. Probably it reflects a high influx of p-coumaric and p-hydroxyphenylpropionic acids (both bacterial metabolites of non-absorbed tyrosine) under exceptional conditions of a heavily loaded β-oxidation by MCT administration.     

Mechanism of action of N-acetylcysteine in the protection against the hepatotoxicity of acetaminophen in rats in vivo

N-Acetylcysteine is the drug of choice for the treatment of an acetaminophen overdose. It is thought to provide cysteine for glutathione synthesis and possibly to form an adduct directly with the toxic metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine. However, these hypothese have not been tested in vivo, and other mechanisms of action such as reduction of the quinoneimine might be responsible for the clinical efficacy of N-acetylcysteine. After the administration to rats of acetaminophen (1 g/kg) intraduodenally (i.d.) and of [³⁵S]-N-acetylcysteine (1.2 g/kg i.d.), the specific activity of the N-acetylcysteine adduct of acetaminophen (mercapturic acid) isolated from urine and assayed by high pressure liquid chromatography averaged 76±6% of the specific activity of the glutathione-acetaminophen adduct excreted in bile, indicating that virtually all N-acetylcysteine-acetaminophen originated from the metabolism of the glutathione-acetaminophen adduct rather than from a direct reaction with the toxic metabolite. N-Acetylcysteine promptly reversed the acetaminophen-induced depletion of glutathione by increasing glutathione synthesis from 0.54 to 2.69 μmol/g per h. Exogenous N-acetylcysteine did not increase the formation of the N-acetylcysteine and glutathione adducts of acetaminophen in fed rats. However, when rats were fasted before the administration of acetaminophen, thereby increasing the stress on the glutathione pool, exogenous N-acetylcysteine significantly increased the formation of the acetaminophen-glutathione adduct from 57 to 105 nmol/min per 100 g. Although the excretion of acetaminophen sulfate increased from 85±15 to 211±17 μmol/100 g per 24 h after N-acetylcysteine, kinetic simulations showed that increased sulfation does not significantly decrease formation of the toxic metabolite. Reduction of the benzoquinoneimine by N-acetylcysteine should result in the formation of N-acetylcysteine disulfides and glutathione disulfide via thiol-disulfide exchange. Acetaminophen alone depleted intracellular glutathione, and led to a progressive decrease in the biliary excretion of glutathione and glutathione disulfide. N-Acetylcysteine alone did not affect the biliary excretion of glutathione disulfide. However, when administered after acetaminophen. N-acetylcysteine produced a marked increase in the biliary excretion of glutathione disulfide from 1.2±0.3 nmol/min per 100 g in control animals to 5.7±0.8 nmol/min per 100 g. Animals treated with acetaminophen and N-acetylcysteine excreted 2.7±0.8 nmol/min per 100 g of N-acetylcysteine disulfides (measured by high performance liquid chromatography) compared to 0.4±0.1 nmol/min per 100 g in rats treated with N-acetylcysteine alone. In conclusion, exogenous N-acetylcysteine does not form significant amounts of conjugate with the reactive metabolite of acetaminophen in the rat in vivo but increases glutathione synthesis, thus providing more substrate for the detoxification of the reactive metabolite in the early phase of an acetaminophen intoxication when the critical reaction with vital macromolecules occurs.     

Purification of three major forms of β-hCG from urine and production of polyclonal antibodies against them

ObjectivesThis study has been conducted to develop a proper and relatively simple method for purification of three major forms of β-hCG from urine to use as clinical calibrators and also to produce anti-β-hCG polyclonal antibodies against these purified forms.Design and methodsMajor β-hCG forms from urine of pregnant women were purified using an ultrafiltration method followed by three successive steps: 1) affinity chromatography on a Concanavalin A-Sepharose column, 2) ion exchange chromatography on a DEAE-Sephacel column and 3) preparative gel electrophoresis. Specific polyclonal antibodies against the purified forms were produced by immunizing rats. We also extracted β-hCG from urine using trypsin affinity chromatography.ResultsThree β-hCG forms with the molecular mass (MW) of 28, 32 and 35 kDa were purified and identified by ELISA method using monoclonal antibodies against two distinct epitopes (β2 and β11) on β-hCG. The titer of prepared antibodies was comparable with common standard anti-β-hCG antibodies. Also we could purify a 37 kDa form of β-hCG by trypsin affinity chromatography.ConclusionsHere we proposed two different proper methods for purification of major forms of β-hCG from urine. We also found that three prepared β-hCG forms expose the β11 epitope and have an hCGβcf epitope expressed. Specific antibodies against purified β-hCG forms had comparable titers and could be used in diagnostic kits.     

高血压伴代谢综合征血、尿β_2-微球蛋白的改变

目的观察高血压伴代谢综合征（metabolic syndrome,MS）患者血、尿β2-微球蛋白（β2-MG）水平的改变,以评价高血压伴MS对早期肾脏功能损害的影响。方法选取2010年11月—2011年10月在我科住院诊断为2级高血压80例作为试验组,按是否伴MS分为两个亚组：单纯高血压组39例;高血压伴MS组41例。另选同期健康体检31例作为对照组。采用同位素放射免疫法测定血、尿β2-MG水平。结果高血压伴MS组血、尿β2-MG水平高于对照组及单纯高血压组（P均〈0.01）;单纯高血压组血、尿β2-MG水平高于对照组（P〈0.05）;血β2-MG水平与空腹血糖、腰围及收缩压呈正相关。结论 MS和高血压均是导致肾功能早期损害的危险因素,高血压伴MS时可加速对肾功能的损害,MS引起早期肾脏损害与多种代谢紊乱因素相关。     

Congenital adrenal hyperplasia. Plasma and urinary steroid conjugates in seven children with steroid 21-hydroxylase deficiency

Plasma neutral steroid sulphates and urinary neutral steroid sulphates and glucuronides from seven children (0.17–10.4 years) with steroid 21-hydroxylase deficiency were determined using gas-liquid chromatography and gas chromatography -mass spectrometry. Three of the patients were salt-losers. Large inter-individual variation in plasma concentration and urinary excretion of these compounds was observed. However, the group did have certain characteristic features. In plasma, the main compounds present were 3β-hydroxy-5-ene steroids. A progesterone metabolite, 5α-pregnane-3β,20α-diol, and a 17α-hydroxyprogesterone metabolite, 5β-pregnane-3α, 17α, 20α-triol, were present as sulphate conjugates and 3α, 17α-dihydroxy-5β-pregnan-20-one sulphate was identified for the first time in human peripheral plasma. In the urine, metabolites of 17α-hydroxyprogesterone were predominant and 5β-pregnane-3α, 17α,20α-triol, excreted mainly as a glucuronide, alone comprised about 50% of the neutral steroid excretion in these subjects. The next most abundant steroid was 3α,17α,20α-trihydroxy-5β-pregnan-11-one. The following compounds have not previously been found in the urine of patients with steroid 21-hydroxylase deficiency but were found in the present subjects: 5-androstene-3β, 17β-diol, 5α-pregnane-3β, 20α-diol and 3β,17α-dihydroxy-5β-pregnan-20-one. The pattern of the plasma and urinary steroids determined clearly differentiates the subjects with a steroid 21-hydroxylase defect from normal subjects and from patients with a 3β-hydroxysteroid dehydrogenase deficiency.     

Redox control of β2‐glycoprotein I–von Willebrand factor interaction by thioredoxin‐1

Summary. Background: β₂‐Glycoprotein I (β₂GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β₂GPI in thrombus formation is unknown. We have recently shown that β₂GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin‐1 and protein disulfide isomerase, and that reduction of β₂GPI can take place on the platelet surface. Methods: β₂GPI, reduced by thioredoxin‐1, was labeled with the selective sulfhydryl probe N^a‐(3‐maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β₂GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin‐activated VWF was studied in the presence of reduced β₂GPI. Results: We demonstrate that the Cys288–Cys326 disulfide in domain V of β₂GPI is the predominant disulfide reduced by thioredoxin‐1. Reduced β₂GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β₂GPI reduced by thioredoxin‐1, in comparison with non‐reduced β₂GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol‐dependent interaction of β₂GPI with VWF may contribute to the redox regulation of platelet adhesion.     

Folic acid non-dependent formiminoglutamic aciduria in two siblings

Two sisters were detected with normal serum folic acid excreting formiminoglutamic acid (FIGLU) in urine in excessive amounts (582 ± 76 mmoles FIGLU/mole creatinine; about 0.5 g/day). The intelligence quotient of the girls is now 0.83 and 1.13, respectively. FIGLU was isolated from urine by ionexchange chromatography, high-voltage electrophoresis and adsorption chromatography on Porapak Q, and was identified by mass spectrometry of the free acid and of the dimethyl ester hydrochloride, by elemental analysis, chemical degradation, and by enzymatic action (EC 2.1.2.5).Increase in serum folate (L. casei) after a single oral dose of folic acid was normal. The urinary FIGLU excretion was not influenced by pharmacological oral doses of folic acid (10 mg/day for 8 days), nor by a load with glycine or serine, but was reduced by a diet low in histidine, and was increased by oral loading with histidine (3 × 198 mg/kg body weight) to about 2900 mmoles/ mole creatinine (about 1.5 g/day). Of the ingested excess histidine, 22% were excreted as FIGLU. Urinary histidine, urocanic acid, formic acid, and aminoimidazole carboxamide remained normal. After a glycine load (0.3 g/kg) serine concentration in serum increased normally.The abnormality is believed to be caused by a folic acid non-dependent deficiency in formimino-l-glutamate :tetrahydrofolate 5-formiminotransferase (EC 2.1.2.5). No liver biopsy was available to measure this enzyme in our subjects. All attempts to measure the enzyme activity in normal human erythrocytes failed. Instead, a significant decrease of hog liver enzyme activity was noted after addition of hemolysate from normal controls.The abnormality differs from the published cases of postulated formiminotransferase deficiency in the 10-fold higher FIGLU excretion after histidine loading, the normal hematological findings, the normal serum folic acid, and the lack of mental retardation in one case.The formation of N,N′-di-(2-glutaryl)formamidine in concentrated solutions of FIGLU is evidenced by mass spectrometry.     

The quantitation of beta-aminoisobutyric acid in urine by mass fragmentography

A specific, rapid and sensitive method for the quantitation of β-aminoisobutyric acid in urine is described. The method is based on the analysis by quadrupole mass fragmentography of its N-TFA-O-n-butyl derivative using α-amino octanoic acid as an internal standard. The procedure is sensitive being able to quantitate as little as 1 ng of β-aminoisobutyric acid and is applicable to the routine analysis of this compound in biological fluids. The analysis time, exclusive of chemical derivatization, occupies about 17 minutes.     

Urinary lysosomal glycosidases after renal allotransplantation: correlation of enzyme excretion with allograft rejection and ischemia

Urinary β-galactosidase, β-glucuronidase and N-acetyl-β-glucosaminidase were measured in patients with renal allotransplants and compared with normal controls. Increased excretion of all three enzymes was noted in the transplant patients resulting possibly from mild chronic rejection.A second part of the investigation correlated renal function with daily N-acetyl-β-glucosaminidase excretion by the patients. In acute rejection, enzyme levels rose sharply from a baseline then decreased following successful treatment. With cadaveric grafts and initially good urinary flow, N-acetyl-β-glucosaminidase levels were high and decreased as creatinine clearance improved; however, with initial oliguria, levels were low and rose as diuresis began then decreased to a baseline. This was attributed to a washing out of enzyme released during the unavoidable ischemic period involved in handling cadaver kidneys.Because it reflects physiological changes in the kidney, daily monitoring of urinary N-acetyl-β-glucosaminidase should be helpful in the diagnosis of renal damage caused by rejection and ischemia.     

Anti-HTLV-I IgG in urine detected by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide,Cys-env gp46(188-224), as antigen

Antibody IgG to human T-cell leukemia virus type I (HTLV-I) in urine was detected by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide, Cys-env gp46(188–224), as antigen, the sensitivity and specificity of which were 100 and 98.5%, respectively, using serum samples. Anti-HTLV-I IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin–Cys-env gp46(188–224) conjugate and Cys-env gp46(188–224)-β-D -galactosidase (Escherichia coli) conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with ?N-2,4-dinitrophenyl-L -lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG γ-chain) IgG. Finally, bound β-D -galactosidase activity was assayed by fluorometry. Thirty-one urine samples from seropositive subjects and 100 urine samples from seronegative subjects were tested. The sensitivity and specificity were 87 and 100%, respectively, with unconcentrated urine samples and 94 and 100%, respectively, with approximately 10-fold concentrated urine samples. These results were superior to those by the conventional ELISA and gelatin particle agglutination test. © 1994 Wiley-Liss, Inc.     

N-Acetyl-L-aspartic acid, N-acetyl-α-l-aspartyl-l-glutamic acid and β-citryl-l-glutamic acid in human urine

$\text{N-}\text{Acetyl-}\text{l}\text{-aspartic}$ acid (NA-Asp), $\text{N-}\text{acetyl}\text{-α-}\text{l}\text{-aspartyl-L-glutamic}$ acid (NA-Asp-Glu) and β-citryl-l-glutamic acid (β-CG), which are known to occur in the brain, have been isolated from human urine. Their identities were proved by comparing them with synthetic NA-Asp, NA-Asp-Glu and β-CG using electrophoretic and Chromatographie methods and by acid hydrolysis.A method was developed for the quantitation of NA-Asp, NA-Asp-Glu and β-CG in human urine. It consists of ion-exchange chromatography followed by gas-chromatographic analysis. The amounts of urinary excretion of NA-Asp, NA-Asp-Glu and ν-CG were 41.2 ± 10.1 (n = 27), 20.8 ± 9.6 (n = 27) and 30.2 ± 13.2 (n = 21) μmol/g creatinine in adult males, and 62.2 ±16.3 (n = 27), 24.0 ±8.2 (n = 27) and 40.5 ± 21.1 (n = 24) μmol/g creatinine in adult females, respectively.     

High-resolution mass spectrometry for thymosins detection and characterization

Objectives: The aim of this study was to characterize β and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies.

Methods: A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides.

Results: In addition to thymosin β₄ (Tβ₄) and β₁₀ (Tβ₁₀), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tβ₄ and Tβ₁₀ together with other proteolytic fragments. The sulfoxide derivative of both Tβ₄ and Tβ₁₀ and the acetylated derivatives at lysine residues of Tβ₄ were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α₁ and thymosin α₁₁ together with diverse proteolytic fragments were identified too.

Conclusion: The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.     

Activity of beta-galactosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in human rectal mucosa

Activity of β-galactosidase, β-glucuronidase and N-acetl-β-glucosaminidase was determined by biopsy specimens of rectal mucosa of control subjects, cystinotic children and their parents.The studied enzymes exhibited maximal activity at pH 5.0, 4.0 and 4.5, respectively. Apparent K_m values using P-nitrophenyl-β-galactoside, p-nitrophenyl-β-glucuronide and p-nitropheny-β-glucosaminide were found to be 0.52 mM, 0.70 mM, and 0.67 mM.The activity of all three enzymes was found to be closely correlated in the 11 subjects of the control group. The values found in parents and their cystinotic children fit into these correlations, but show higher scatter of data caused by the fact that values of β-galactosidase were found to be higher and of β-glucuronidase and N-acetyl-glucosaminidase lower in the group of parents than in the other two groups.     

Determination of 3-mercaptolactate, mercaptoacetate and N-acetylcysteine in urine by gas chromatography

A method is described for the determination of 3-mercaptolactate, mercaptoacetate (thioglycolate) and N-acetylcysteine in urine. As these compounds are mainly excreted as their mixed disulfides with cysteine, they are first reduced to the free thiols by an insoluble polymer containing thiol groups. After purification by chromatography on an organomercurial adsorbent, the compounds are converted to benzyl derivatives by extractive alkylation and determined by gas chromatography. The identity of the compounds analyzed was verified by mass spectrometry. It was demonstrated that mercaptolactate and mercaptoacetate are almost entirely excreted as their mixed disulfides with cysteine, whereas appreciable amounts of N-acetylcyteine are present as the symmetrical disulfide and the free thiol. The urinary excretion of the compounds from healthy human beings was also studied.     

β2- and β3-adrenergic receptors drive COMT-dependent pain by increasing production of nitric oxide and cytokines

Decreased activity of catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, contributes to pain in humans and animals. Previously, we demonstrated that development of COMT-dependent pain is mediated by both β₂- and β₃-adrenergic receptors (β₂ARs and β₃ARs). Here we investigated molecules downstream of β₂- and β₃ARs driving pain in animals with decreased COMT activity. Based on evidence linking their role in pain and synthesis downstream of β₂- and β₃AR stimulation, we hypothesized that nitric oxide (NO) and proinflammatory cytokines drive COMT-dependent pain. To test this, we measured plasma NO derivatives and cytokines in rats receiving the COMT inhibitor OR486 in the presence or absence of the β₂AR antagonist ICI118,551 + β₃AR antagonist SR59320A. We also assessed whether the NO synthase inhibitor L-N_G-nitroarginine methyl ester (L-NAME) and cytokine-neutralizing antibodies block the development of COMT-dependent pain. Results showed that animals receiving OR486 exhibited higher levels of NO derivatives, tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2) in a β₂- and β₃AR-dependent manner. Additionally, inhibition of NO synthases and neutralization of the innate immunity cytokines TNFα, IL-1β, and IL-6 blocked the development of COMT-dependent pain. Finally, we found that NO influences TNFα, IL-1β, IL-6, and CCL2 levels, whereas TNFα and IL-6 influence NO levels. Altogether, these results demonstrate that β₂- and β₃ARs contribute to COMT-dependent pain, at least partly, by increasing NO and cytokines. Furthermore, they identify β₂- and β₃ARs, NO, and proinflammatory cytokines as potential therapeutic targets for pain patients with abnormalities in COMT physiology.     

Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders.In amniotic fluid β-galactosidase and α-mannosidase decreased moderately towards term, while β-glucosidase decreased markedly. N-Acetyl-β-glucosaminidase and β-glucuronidase were relatively unchanged.In chorionic villi N-acetyl-β-glucosaminidase, β-galactosidase, and α-mannosidase were substantially decreased towards term, while β-glucosidase was unchanged and β-glucuronidase markedly increased.In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study.The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.     

Prothiofos Metabolites in Human Poisoning

Case Report: A 63-year-old woman was admitted to a local hospital after the ingestion of 40% prothiofos preparation (Tokuthion®) 370 mL. Gastric lavage was performed and cathartics, active charcoal, diuretics, atropine sulfate, and pralidoxime were administered. Serum cholinesterase activity was 1.3 IU/L (normal 200–460 IU/L). The patient's consciousness was gradually restored after 4 hours of charcoal hemoperfusion and she was discharged 5 days after admission with no sequelae. Method: Plasma and urine prothiofos and metabolites were detected by gas chromatography–flame photometry and gas chromatography–mass spectrometry. Two despropyl metabolites were synthesized for identification and estimation. Results: The main metabolites were identified with authentic prothiofos and methyl esters of synthesized des-S-propyl prothiofos oxon (O-2,4-dichlorophenyl O-ethyl phosphate), despropyl prothiofos oxon (O-2,4-dichlorophenyl O-ethyl phospholothiolate), and des-S-propyl prothiofos (O-2,4-dichlorophenyl O-ethyl phosphorothioate). Despropyl prothiofos (O-2,4-dichlorophenyl O-ethyl phosphorodithioate) was also identified in plasma. Large amounts of the hydrolyzed product, 2,4-dichlorophenol and its conjugate were also found. The metabolic pattern of prothiofos in humans appears to be different from that in rats.