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.

Total purine and purine base content of common foodstuffs for facilitating nutritional therapy for gout and hyperuricemia.

Purines are natural substances found in all of the body's cells and in virtually all foods. In humans, purines are metabolized to uric acid, which serves as an antioxidant and helps to prevent damage caused by active oxygen species. A continuous supply of uric acid is important for protecting human blood vessels. However, frequent and high intake of purine-rich foods reportedly enhances serum uric acid levels, which results in gout and could be a risk factor for cardiovascular disease, kidney disease, and metabolic syndrome. In Japan, the daily intake of dietary purines is recommended to be less than 400 mg to prevent gout and hyperuricemia. We have established an HPLC method for purine analysis and determined purines in a total of 270 foodstuffs. A relatively small number of foods contained concentrated amounts of purines. For the most part, purine-rich foods are also energy-rich foods, and include animal meats, fish meats, organs such as the liver and fish milt, and yeast. When the ratio of the four purine bases (adenine, guanine, hypoxanthine, and xanthine) was compared, two groups of foods were identified: one that contained mainly adenine and guanine and one that contained mainly hypoxanthine. For patients with gout and hyperuricemia, the amount of total purines and the types of purines consumed, particularly hypoxanthine, are important considerations. In this context, the data from our analysis provide a purine content reference, and thereby clinicians and patients could utilize that reference in nutritional therapy for gout and hyperuricemia.

Proteomic analysis of a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate: a case report.

The objective of the present study was to investigate the matrix protein of a rare urinary stone that contained calcium carbonate. A urinary stone was extracted from a 34-year-old male patient with metabolic alkalosis. After X-ray diffractometry and infrared analysis of the stone, proteomic analysis was carried out. The resulting mass spectra were evaluated with protein search software, and matrix proteins were identified. X-ray diffraction and infrared analysis confirmed that the stone contained calcium carbonate and calcium oxalate dihydrate. Of the identified 53 proteins, 24 have not been previously reported from calcium oxalate- or calcium phosphate-containing stones. The protease inhibitors and several proteins related to cell adhesion or the cytoskeleton were identified for the first time. We analyzed in detail a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate. Considering the formation of a calcium carbonate stone, the new identified proteins should play an important role on the urolithiasis process in alkaline condition.

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.

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).

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 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.

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%.

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).

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.

Detecting Abnormal Word Utterances in Children With Autism Spectrum Disorders: Machine-Learning-Based Voice Analysis Versus Speech Therapists.

Abnormal prosody is often evident in the voice intonations of individuals with autism spectrum disorders. We compared a machine-learning-based voice analysis with human hearing judgments made by 10 speech therapists for classifying children with autism spectrum disorders ( n = 30) and typical development ( n = 51). Using stimuli limited to single-word utterances, machine-learning-based voice analysis was superior to speech therapist judgments. There was a significantly higher true-positive than false-negative rate for machine-learning-based voice analysis but not for speech therapists. Results are discussed in terms of some artificiality of clinician judgments based on single-word utterances, and the objectivity machine-learning-based voice analysis adds to judging abnormal prosody.

Joint Attention Development in Low-risk Very Low Birth Weight Infants at Around 18 Months of Age.

The purpose of this study was to clarify the developmental characteristics of joint attention in very low birth weight (VLBW) infants with a low risk of complications. Section B of the Checklist for Autism in Toddlers (CHAT) was administered to 31 VLBW and 45 normal birth weight (NBW) infants aged 18-22 months, while the sessions were recorded with a video camera. A semi-structured observation scale was developed to assess infants' joint attention from the video footage, and was shown to be reliable. VLBW, compared to NBW, infants showed significantly poorer skills in 2 of 4 items on responding to joint attention, and in 6 of 10 items on initiating joint attention. VLBW infants need more clues in order to produce joint attention. The difficulty was attributed to insufficient verbal and fine motor function skills. Continuous follow-up evaluation is essential for both high-risk and low-risk VLBW infants and their parents.

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.

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.

Proteomic analysis to examine the role of matrix proteins in a gouty tophus from a patient with recurrent gout.

To examine the role of matrix proteins in the formation of gouty tophus, we analyzed the crystalline components and matrix proteins in a gouty tophus from a patient with recurrent gout. Micro-area X-ray diffraction analysis and infrared spectroscopy indicated that the tophus was composed of monosodium urate monohydrate. Proteomic analysis identified 134 proteins from the tophus as matrix proteins. Many proteins relevant to inflammation and host defense were identified, and immunoglobulin was detected in all four extracted fractions (KCl, formic acid, guanidine-HCl, and ethylenediaminetetraacetic acid) and from many spots throughout a broad molecular weight range after electrophoresis. It is thought that the process of biological defense including the immunity has occurred in the gouty tophus.

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system.

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid-liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30-1000ng for nicotine and 0.06-1000ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8-15ng/mL and 156-372ng/mL, respectively.

A simple HPLC method for determining the purine content of beer and beer-like alcoholic beverages.

Several methods for quantifying the purine content in food and drink have been described using high-performance liquid chromatography (HPLC). We have developed an improved HPLC method that is based on a method reported by Kaneko et al. and that is more sensitive yet simple, and suitable for determining the purine content of beer and beer-like alcoholic beverages. Quantitative HPLC separation was performed on a Shodex Asahi Pak GS-320HQ column with an isocratic elution of 150 mmol/L sodium phosphate buffer (H(3)PO(4)/NaH(2)PO(4) = 20:100 (v/v)). The retention times for the four analytes, namely, adenine, guanine, hypoxanthine and xanthine, were 19.9, 25.0, 29.3 and 43.0 min, respectively. The resolution was good, and there was no excessive interference from the other compounds in the beverages at these retention times. Furthermore, the detection limit for all the analytes was improved to less than 0.0075 mg/L, and all the calibration curves showed good linearity (r(2) > 0.999) between 0.013 and 10 mg/L for adenine and guanine, and between 0.025 and 10 mg/L for hypoxanthine and xanthine. The pretreatment was simplified by removing some procedures and optimizing the perchloric acid hydrolysis and the enzymatic peak-shift assay. We reduced the sample dilution rate by almost 50%, and the time spent on pretreatment from 4 days to only 180 min. The recovery of the analytes from spiked samples was 94.8 - 103.8%. This method may be useful for evaluating quantitative and qualitative differences in the purine content of beer and beer-like alcoholic beverages.

Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA.

N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions and is oxidized by an equimolar amount of H2O2 to yield its decarboxylated product 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid (ADOA). Highly sensitive analytical methods are required to detect ADOA in the human body. We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) to enable their fluorometric detection, and developed a method using HPLC with fluorometric detection (HPLC-FD) for the simultaneous determination of the derivatized NANA and ADOA. The derivatized NANA and ADOA were separated by a hydrophilic interaction liquid chromatography (HILIC) column using an H2O/CH3CN/HCOOH (10/90/0.35) mobile phase. Fluorescence was monitored at excitation and emission wavelengths of 450nm and 560nm, respectively. Both intra- and inter-day (n=6) repeat determinations of the DBD-ED-derivatized NANA and ADOA gave relative standard deviations of less than 5%. The calibration curves for standard solutions of DBD-ED-derivatized NANA and ADOA were linear over the ranges from 576fmol to 2.0nmol and 556fmol to 2.0nmol, respectively. The method developed was highly specific and sensitive for NANA and ADOA. The presence of ADOA in biological samples was revealed for the first time using this method.