Plasma levels and urinary excretion of protein Z in patients with urolithiasis.

OBJECTIVES: Protein Z (PZ) is a γ-carboxyglutamic acid protein present in plasma that is involved in blood coagulation. Detailed analysis of urinary stones from patients with urolithiasis has revealed that PZ is often found in urinary stones composed of calcium oxalate monohydrate. In this study, we compared blood and urinary PZ concentrations between healthy individuals and patients with urolithiasis. METHODS: Plasma and urine were collected from healthy individuals and patients with urolithiasis who provided informed consent. PZ was detected as a urinary stone matrix protein in some of the patients. PZ was quantified by ELISA, creatinine was measured by the enzymatic method, and the total protein concentration was measured by the Bradford method. RESULTS: The plasma PZ level was 2.54 ± 1.02 µg/mL in healthy individuals and that in urolithiasis patients classified by stone history were from 1.16 ± 0.77 to 3.73 ± 1.09 µg/mL, which was not significantly different. The urinary excretion of PZ (PZ/creatinine) was also not different in patients with urolithiasis and in healthy individuals (from 54.1 ± 40.9 to 95.4 ± 69.4 ng/mg vs. 73.3 ± 36.0 ng/mg). A positive correlation was found between the plasma PZ level and creatinine-corrected urinary PZ concentration (r = 0.46). CONCLUSIONS: Both the plasma level and urinary excretion of PZ in urolithiasis patients were not significantly different with normal individuals. PZ detected in urinary stones as a matrix protein is thought to be incorporated into urinary stones regardless of blood and urine levels of PZ.

Determination of Picolinic Acid by HPLC Coupled With Postcolumn Photo Irradiation Using Zinc Acetate as a Fluorescent Derivatization Reagent.

For the fluorometric determination of picolinic acid in human serum, HPLC-postcolumn UV irradiation using zinc acetate has been developed. Picolinic acid in serum sample was separated on a Capcell Pak C18. The mobile phase consisted of 0.1 mol/L sodium phosphate solution (adjusted to pH 3.0) containing 3.0 mmol/L zinc acetate and 3.5 mmol/L trimethylamine, and delivered at a flow rate of 0.8 mL/minutes. In order to stabilize the retention time (6.5 minutes), a back pressure tube (0.4 m × 0.13 mm i.d.) was attached after the photoreaction tube. Column effluent was irradiated with ultraviolet light to produce fluorescence, excitation wavelength of 336 nm and emission wavelength of 448 nm. The calibration graph for picolinic acid showed linearity when the amount was in the range of 0.89 to 455 pmol, and the detection limit (S/N = 3) was determined to be 0.30 pmol. The pretreatment of serum sample consisted of deproteinized by perchloric acid, potassium hydroxide, and mobile phase. The mean recovery of picolinic acid from serum was 99.0%. Using this procedure, the concentration of picolinic acid in serum of a healthy subject was determined.

Possible contribution of phosphate to the pathogenesis of chronic kidney disease in dolphins.

This study aimed to investigate whether phosphate contributes to the pathogenesis of chronic kidney disease (CKD) in dolphins. Renal necropsy tissue of an aged captive dolphin was analyzed and in vitro experiments using cultured immortalized dolphin proximal tubular (DolKT-1) cells were performed. An older dolphin in captivity died of myocarditis, but its renal function was within the normal range until shortly before death. In renal necropsy tissue, obvious glomerular and tubulointerstitial changes were not observed except for renal infarction resulting from myocarditis. However, a computed tomography scan showed medullary calcification in reniculi. Micro area X-ray diffractometry and infrared absorption spectrometry showed that the calcified areas were primarily composed of hydroxyapatite. In vitro experiments showed that treatment with both phosphate and calciprotein particles (CPPs) resulted in cell viability loss and lactate dehydrogenase release in DolKT-1 cells. However, treatment with magnesium markedly attenuated this cellular injury induced by phosphate, but not by CPPs. Magnesium dose-dependently decreased CPP formation. These data support the hypothesis that continuous exposure to high phosphate contributes to the progression of CKD in captive-aged dolphins. Our data also suggest that phosphate-induced renal injury is mediated by CPP formation in dolphins, and it is attenuated by magnesium administration.

Purine content of hospital meals and its effect on serum uric acid, urine pH, and urinary uric acid excretion.

Hospital meals are prepared with the nutrients required by the patient's medical condition in consideration. However, no research on the purine content of hospital meals has been conducted, and it is not shown on the purine content. The recommended purine consumption for patients with gout and hyperuricemia is 400 mg/day based on the Japanese guidelines for the management of hyperuricemia and gout. In this study, the purine content in hospital meals was evaluated using the purine content of foods previously determined by our laboratory as a reference. The serum uric acid levels and uric acid excretion in admitted patients who consumed these diets were examined.

Determination of total purine and free purine content in milk, soymilk, and enteral nutritional supplements to assist nutritional therapy for hyperuricemia and gout.

In this study, we determined the purine contents in milk and soymilk, as protein-rich drinks, and in enteral nutritional supplements employed to ameliorate protein malnutrition in the elderly. Milk consumption is known to lower serum uric acid levels and to promote uric acid excretion. However, discrepant results have been reported regarding the effect of soymilk on serum uric acid levels. The purpose of this study was to quantify and compare the total purine contents and the contents of individual purines and pyrimidines by molecular type (nucleotides, nucleosides, and bases).

Xanthinuria Type 1 with a Novel Mutation in Xanthine Dehydrogenase and a Normal Endothelial Function.

Whether or not extremely low levels of serum uric acid (SUA) in xanthinuria are associated with impairment of the endothelial function and exercise-induced acute kidney injury (EIAKI) is unclear. A 59-year-old woman without EIAKI or urolithiasis had undetectable levels of UA in serum and urine and elevated levels of hypoxanthine and xanthine in urine. A genetic analysis revealed homozygous mutations in the XDH gene [c.1585 C>T (p. Gln529*)]. Flow-mediated dilation was within the normal range. This is the first report of a case with extremely low levels of SUA, xanthinuria with novel mutations of xanthine dehydrogenase (XDH) and a normal endothelial function.

Increased Lung Uric Acid Deteriorates Pulmonary Arterial Hypertension.

Background Recent studies have demonstrated that uric acid (UA) enhances arginase activity, resulting in decreased NO in endothelial cells. However, the role of lung UA in pulmonary arterial hypertension (PAH) remains uncertain. We hypothesized that increased lung UA level contributes to the progression of PAH. Methods and Results In cultured human pulmonary arterial endothelial cells, voltage-driven urate transporter 1 (URATv1) gene expression was detected, and treatment with UA increased arginase activity. In perfused lung preparations of VEGF receptor blocker (SU5416)/hypoxia/normoxia-induced PAH model rats, addition of UA induced a greater pressure response than that seen in the control and decreased lung cGMP level. UA-induced pressor responses were abolished by benzbromarone, a UA transporter inhibitor, or L-norvaline, an arginase inhibitor. In PAH model rats, induction of hyperuricemia by administering 2% oxonic acid significantly increased lung UA level and induced greater elevation of right ventricular systolic pressure with exacerbation of occlusive neointimal lesions in small pulmonary arteries, compared with nonhyperuricemic PAH rats. Administration of benzbromarone to hyperuricemic PAH rats significantly reduced lung UA levels without changing XOR (xanthine oxidoreductase) activity, and attenuated right ventricular systolic pressure increase and occlusive lesion development. Topiroxostat, a XOR inhibitor, significantly reduced lung XOR activity in PAH rats, with no effects on increase in right ventricular systolic pressure, arterial elastance, and occlusive lesions. XOR-knockout had no effects on right ventricular systolic pressure increase and arteriolar muscularization in hypoxia-exposed mice. Conclusions Increased lung UA per se deteriorated PAH, whereas XOR had little impact. The mechanism of increased lung UA may be a novel therapeutic target for PAH complicated with hyperuricemia.

Effects of islatravir (4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA) on renal tubular cells and islatravir's interactions with organic anion transporters.

Islatravir (ISL; 4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA) is a novel reverse transcriptase translocation inhibitor and has a unique structure and high antiviral activity against wild-type and multidrug resistant HIV strains. In this study, we investigated whether islatravir (ISL) can cause kidney damage compared to tenofovir disoproxil fumarate (TDF) and tenofovir (TFV). We also investigated interactions of these drugs with organic anion transporters (OATs). There is a large gap in ISL concentration between the pharmacological dose to proximal tubular cells and the clinical dose. ISL is unlikely to be taken up via OAT1 or OAT3; therefore, OAT1 and OAT3 may not be involved in the injury to tubular cells. Present data strongly suggests that ISL is not toxic to proximal tubules because blood levels of ISL are not high enough to cause kidney damage in the clinical setting.

Differentiation of Positional Isomers of Halogenated Benzoylindole Synthetic Cannabinoid Derivatives in Serum by Hybrid Quadrupole/Orbitrap Mass Spectrometry.

Legally regulated synthetic cannabinoids (SCs) are continuously being created by making minor positional modifications to pre-existing analogs; thus, compounds with minor structural differences must be isolated and identified accurately. For iodo-benzoylindole derivatives of SCs, only specific isomers are currently the target of legal control, and it is necessary to establish an analytical method for accurately identifying positional isomers. In this study, we synthesized a series of 57 designer drugs and developed a screening method for identifying halogen positional isomers on the phenyl ring of benzoylindole derivative SCs in serum. Analytical methods using the Discovery F5 pentafluorophenyl column gave the best selectivity and retention of the positional isomer analytes. Some of the meta and para iodo-substituted SCs were eluted at similar retention times and were difficult to separate by liquid chromatography (LC). However, they were identified via the relative abundance of the two product ions in the collision-induced dissociation reaction using LC-hybrid quadrupole/orbitrap high-resolution mass spectrometry. Our synthesized halogen-substituted positional isomer SC library and method for differentiating positional isomers of halogenated benzoylindole SC derivatives could provide an indispensable analysis tool for identifying illegal drugs in serum of drug users.

Determination of total purine and purine base content of 80 food products to aid nutritional therapy for gout and hyperuricemia.

The aim of this work is to facilitate the nutritional therapy of gout and hyperuricemia. In Japan, patients with gout or hyperuricemia are recommended to consume less than 400 mg of dietary purines per day. When receiving nutritional therapy for gout or hyperuricemia, purine-rich foods (>200 mg/100 g) should be eaten in even lower quantities. The purine content of foods reported in this study are as follows: noodles, 0.6-12.1 mg/100 g; bread, 4.4 mg/100 g; peas or seeds, 19.6-67.1 mg/100 g; dairy, 0.0-1.4 mg/100 g; Japanese vegetables, 0.9-47.1 mg/100 g; seasonings, 0.7-847.1 mg/100 g; meat or fish, 19.0-385.4 mg/100 g; fish milt, 375.4-559.8 mg/100 g; and supplements, 81.9-516.0 mg/100 g. Foods containing very large amounts of purine (>300 mg/100 g) included anchovy, cutlassfish (hairtail), cod milt, globefish milt, dried Chinese soup stock, dried yeast, a Euglena supplement, and a Lactobacillus supplement. When eating these high-purine food or supplements, the quantity taken at one meal should be limited, especially milt because they typically consumed amount of 20-30 g is equivalent to 75-168 mg total purines. This is 20%-40% of the recommended daily amount (400 mg/day) for patients with gout or hyperuricemia. Thus, these patients should restrict the amount of purine-rich foods they consume. Good dietary habits with a good balance of nutrients are recommended.

Determination of Dipicolinic Acid in "Natto" by High-Performance Liquid Chromatography Coupled With Postcolumn Photoirradiation With Zinc Acetate.

A system was developed for determining dipicolinic acid in "natto" using liquid chromatography with fluorometric detection. The compound was separated by reversed-phase chromatography using a mobile phase of 0.1 mol/L disodium hydrogen phosphate, 0.05 mol/L citric acid buffer (adjusted to pH 3.0) containing 3.0 mmol/L zinc acetate and 35 mmol/L perchloric acid. The compound in the column effluent was irradiated with ultraviolet light to produce fluorescence. This fluorescence was monitored at an excitation at 336 nm and an emission at 448 nm. The calibration curve for dipicolinic acid was observed to be linear in a range of 0.2 to 112 ng. The dipicolinic acid content of natto was 7.24 ± 0.54 mg/100 g (wet weight, mean ± standard deviation [SD], n = 6).

Simultaneous Determination of Kynurenine and Kynurenic Acid by High-Performance Liquid Chromatography Photoirradiation System Using a Mobile Phase Containing 18-crown-6.

A high-performance liquid chromatography (HPLC) system has been developed for the fluorometric determination of kynurenine (KYN) and kynurenic acid (KYNA) in human serum using a mobile phase containing 18-crown-6. A retention time of KYNA was adjusted with pH of phosphate buffer in 18-crown-6. KYN and KYNA were separated on a CAPCELLPAK C18 (250 × 4.6 mm i.d.). The mobile phase consisted of 35 mmol/L phosphate buffer (pH 8.0)/methanol (85/15, v/v) containing 35 mmol/L hydrogen peroxide and 10 mmol/L 18-crown-6. The retention times of KYN and KYNA were 18and 24 minutes, respectively. The calibration graphs of KYN and KYNA were linear over the range 180 to 2900 and 1 to 84 nmol/L by injecting a 50-µL volume of KYN and KYNA, respectively. Pretreatment of serum was achieved by deproteinization only. The mean recoveries of KYN and KYNA from serum were more than 97%.

Trial analysis of drug profiling by liquid chromatography/mass spectrometry and inductively coupled plasma mass spectrometry to establish the origin of ephedrines used as precursors for illicit production of methamphetamine.

RATIONALE: We investigated whether chemical information on the origin of ephedrine and pseudoephedrine (ephedrines) can be acquired by liquid chromatography/mass spectrometry (LC/MS) as a substitute method for stable isotope ratio mass spectrometry (IRMS), which is not routinely available in forensic laboratories. We examined the characteristic inorganic elemental contaminants of ephedrines as a preliminary study. METHODS: The stable isotope ratios measured by IRMS analysis are expressed relative to the stable isotope ratios of conventional standards. Referring to the method using validated standard samples in IRMS, we selected a standard sample for acquiring stable isotopic ratio by LC/MS. The abundance ratio of the [M + 2H]+ ion to the [M + H]+ ion was measured by means of selected ion monitoring. We carried out qualitative analysis of inorganic elements contained in ephedrines produced by different manufacturing methods with ICPMS. RESULTS: We found that the ratio of stable isotope ion to molecular ion (stable isotope ratio) of ephedrines could be measured with LC/MS. The stable isotope ratio of ephedrines determined by LC/MS were confirmed to show relatively good correlations with the carbon and hydrogen stable isotope ratios found by IR-MS. We identified strontium as a characteristic inorganic element contained in ephedrines prepared by the semisynthetic method from molasses, or in the biosynthetic method from ephedra plants. CONCLUSIONS: Our results suggest that useful chemical information can be obtained by LC/MS, which is easy to carry out, and is generally available in forensic laboratories. It would be worthwhile to investigate the usefulness of stable isotope ratio measurements of Sr in the future.

Proteomic analysis of a urinary stone with two layers composed of calcium oxalate monohydrate and uric acid.

We examined the mechanism of urinary stone formation by analyzing the matrix proteins in a urinary stone with two layers composed of different crystals. Micro-area X-ray spectrometry and infrared spectroscopy revealed calcium oxalate monohydrate in the outside and uric acid in the inside. We also examined the interface. After the outside, inside, and interface parts were separated, proteomic analysis identified 48, 7, and 4 matrix proteins, respectively. Urinary stones with two layers are considered to have grown under different conditions. The matrix proteins in each part differed among the crystal components and may reveal the stone-generating process. The proteins in the interface likely function to enlarge the stone via the addition of different crystals.

Sensitive Analysis of Sialic Acid and Related Compound by Hydrophilic Interaction Liquid Chromatography Using Fluorescence Detection after Derivatization with DBD-PZ.

N-Acetylneuraminic acid (NANA) has been reported to react with hydrogen peroxide in vitro to produce 4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (ADOA). We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) for simultaneous detection. The derivatized NANA and ADOA were separated using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. The calibration curves of DBD-PZ-derivatized NANA and ADOA showed good linearity in the range of 221 fmol to 1.5 nmol, and 44 fmol to 1.5 nmol, respectively. This analytical method has high specificity and is useful for the detection of NANA and ADOA in saliva and serum.

Analysis of l-DOPA-derived melanin and a novel degradation product formed under alkaline conditions.

When the therapeutic drug l-DOPA, which is used to treat Parkinson's disease, is combined with magnesium oxide (MgO), a formulation change produces a dark substance. Infrared spectroscopy reveals that this substance is melanin. After allowing the l-DOPA and MgO mixture to stand, the l-DOPA content decreases significantly, and a new degradation product (the final degradation product of l-DOPA, FDP-D) is generated. Formation of this product requires a solution with a pH of >10, and the presence of MgO is not necessary. FDP-D is not produced by tyrosinase decomposition of l-DOPA and is therefore not a melanin-related compound. Pure FDP-D is isolated by adjusting the l-DOPA solution to pH 10 with ammonium hydroxide, allowing it to stand for 3 days at room temperature, adding trifluoroacetic acid (TFA), filtering the precipitate, and separating the supernatant with high-performance liquid chromatography (HPLC). Mass spectrometry indicates that the isolated FDP-D has a molecular formula of C9H9NO7. On the basis of NMR analysis ((1)H NMR, (13)C NMR, DEPT, H-H COSY, HMQC, and HMBC), FDP-D appears to be a substance with the novel structure 7a-hydroxy-5-oxo-1,2,3,5,7,7a-hexahydropyrano [3,4-b]pyrrole-2,7-dicarboxylic acid.

Targeting Uric Acid and the Inhibition of Progression to End-Stage Renal Disease--A Propensity Score Analysis.

BACKGROUND: The role of uric acid (UA) in the progression of chronic kidney disease (CKD) remains controversial due to the unavoidable cause and result relationship. This study was aimed to clarify the independent impact of UA on the subsequent risk of end-stage renal disease (ESRD) by a propensity score analysis. METHODS: A retrospective CKD cohort was used (n = 803). Baseline 23 covariates were subjected to a multivariate binary logistic regression with the targeted time-averaged UA of 6.0, 6.5 or 7.0 mg/dL. The participants trimmed 2.5 percentile from the extreme ends of the cohort underwent propensity score analyses consisting of matching, stratification on quintile and covariate adjustment. Covariate balances after 1:1 matching without replacement were tested for by paired analysis and standardized differences. A stratified Cox regression and a Cox regression adjusted for logit of propensity scores were examined. RESULTS: After propensity score matching, the higher UA showed elevated hazard ratios (HRs) by Kaplan-Meier analysis (≥ 6.0 mg/dL, HR 4.53, 95%CI 1.79-11.43; ≥ 6.5 mg/dL, HR 3.39, 95%CI 1.55-7.42; ≥ 7.0 mg/dL, HR 2.19, 95%CI 1.28-3.75). The number needed to treat was 8 to 9 over 5 years. A stratified Cox regression likewise showed significant crude HRs (≥ 6.0 mg/dL, HR 3.63, 95%CI 1.25-10.58; ≥ 6.5 mg/dL, HR 3.46, 95%CI 1.56-7.68; ≥ 7.0 mg/dL, HR 2.05, 95%CI 1.21-3.48). Adjusted HR lost its significance at 6.0 mg/dL. The adjustment for the logit of the propensity scores showed the similar results but with worse model fittings than the stratification method. Upon further adjustment for other covariates the significance was attained at 6.5 mg/dL. CONCLUSIONS: Three different methods of the propensity score analysis showed consistent results that the higher UA accelerates the progression to the subsequent ESRD. A stratified Cox regression outperforms other methods in generalizability and adjusting for residual bias. Serum UA should be targeted less than 6.5 mg/dL.

Analysis of Intra- and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography.

To evaluate cellular uptake and purine transport, we developed a high-performance liquid chromatography method for intra- and extracellular purine quantification. Our aim was to develop an effective method for simultaneously quantifying the substrate and metabolites with high sensitivity. C18 columns from different manufacturers were tested for simultaneous quantification of 22 different purine bases, nucleosides, and nucleotides. We used a YMC-Triart C18 column. The analysis conditions, including extraction solutions for the cells and cell culture medium, were optimized to achieve good quantification. Linearity, accuracy, determination limits, and recovery were assessed and showed good performance. The developed HPLC method was successfully applied to the qualitative analysis of 22 different intra- and extracellular purines, demonstrating that it is useful for studying the overall pattern of purine metabolism. This method could also be useful for evaluating metabolic dynamics of purines under a variety of stimulatory conditions of culture cells.

Proteomic Analysis after Sequential Extraction of Matrix Proteins in Urinary Stones Composed of Calcium Oxalate Monohydrate and Calcium Oxalate Dihydrate.

In this study, we performed proteomic analysis following sequential protein extraction on calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) urinary stones to determine the specific matrix proteins according to the crystal components of the stones. After X-ray and IR analysis of 13 urinary stones, matrix proteins were sequentially extracted with KCl, formic acid, guanidine-HCl, and EDTA, before SDS-electrophoresis followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The electrophoretic patterns of the extracted proteins differed from that of COM and COD stones. LC-MS/MS identified 65 proteins, of which many were cellular plasma proteins, and were frequently detected regardless of the crystal components. However, 6 proteins (protein Z, protein S, prothrombin, osteopontin, fatty acid binding protein 5, and ubiquitin) were detected in the final EDTA fractions of COM stones. These proteins are involved in the coagulation process or osteometabolism, and thus the roles they play are of particular interest.