Tomoyo Yamanobe

Determination of purine contents of alcoholic beverages using high performance liquid chromatography.

The purine contents of alcoholic beverages were determined in order to utilize them in the dietary care of gout and hyperuricemia. In the management of these diseases, restriction of both alcohol and purine intake are important. The method employed in this study is a quantitative determination of purine contents by HPLC. Alcoholic beverages were hydrolyzed to corresponding purine bases, which were then separated by HPLC, and base peaks were identified using an enzymatic peak-shift technique. This method is sufficiently accurate and reproducible to examine the purine contents of various alcoholic beverages that patients consume. Purine contents were as follows: spirits, 0.7-26.4 micromol/L; regular beer, 225.0-580.2 micromol/L; low-malt beer, 193.4-267.9 micromol/L; low-malt and low-purine beer, 13.3 micromol/L; other liquors, 13.1-818.3 micromol/L. Some local and low-alcohol beers were found to contain about 2.5 times more purines than regular beer. As some alcoholic beverages contain considerable amounts of purines, we recommend that excess consumption of these beverages be avoided. These data should be useful in the management of hyperuricemia and gout, not only for patients but also for physicians.

Comparison of matrix proteins in different types of urinary stone by proteomic analysis using liquid chromatography–tandem mass spectrometry

Objectives:  To analyze the crystal components and matrix proteins of urinary stones by proteomic analysis using liquid chromatography–tandem mass spectrometry.

Analysis of urinary calculi obtained from a patient with idiopathic hypouricemia using micro area x-ray diffractometry and LC-MS

Urolithiasis is a common complication in patients with hypouricemia. Using a microarea x-ray diffractometer and nanoflow liquid chromatography-mass spectrometry (LC-MS) following SDS-polyacrylamide gel electrophoresis (PAGE), recurrent urinary calculi complicating a hypouricemic patient were analyzed. Analysis with the microarea x-ray diffractometer showed that one of the calculi was composed of calcium oxalate monohydrate and hydroxyapatite. The other was found to be formed from calcium oxalate dihydrate. After determination with LC-MS, both were found to contain uromodulin, albumin, osteopontin, protein Z, and defensins. Lysozyme and calgranulin A were also identified in these calculi. Defensins, which were antimicrobial peptides, and lysozyme, a mucopeptide glycohydrolase, were identified as new organic components of urinary stones. The role of these proteins in the process of urolithiasis is of particular interest.

Purine contents of soybean-derived foods and selected Japanese vegetables and mushrooms.

Purine contents of soybean-derived food and various other Japanese foods were quantitatively determined by high-performance liquid chromatography (HPLC). Purine contents were as follows: soybean-derived foods, 21.9–172.5 mg/100 g or 100 mL; Japanese vegetables, 2.3–171.8 mg/100 g; Japanese mushrooms, 9.5–142.3 mg/100 g. Since purine levels in these foods did not exceed 200 mg/100 g, we recommend that eating of them should be adopted and good dietary habits followed.

A new membrane-associated Ca2+-binding protein of rat spermatogenic cells: Its purification and characterization

A Ca(2+)-binding protein of Mr = 52000, estimated by SDS-PAGE, was purified to a final yield of 0.04% from rat spermatogenic cells. Purification steps included gel filtration, ammonium sulfate precipitation and HPLC. Amino acid analysis showed the content of 34% acidic residues and 15% basic residues. The isoelectric point of this protein was 4.7. Dot-blot analysis indicated that the Ca(2+)-binding protein bound 2 mol of calcium per mol of protein. This protein had two binding sites with dissociation constants of 4.8 microM and 0.2 microM. No appreciable amount of hexose was observed (less than 1 microgram of hexose/70 micrograms of protein). This protein may play an important role such as the Ca(2+)-transport in the plasma membrane of spermatogenic cells.

Urinary Stone Analysis in a Patient with Hyperuricemia to Determine the Mechanism of Stone Formation

In order to determine the mechanism of urinary stone formation in patients with hyperuricemia, we analyzed the crystal components and matrix proteins in a urinary stone from such a patient. Micro-area X-ray spectrometry and infrared (IR) spectroscopy suggested that the outside of the stone was composed of calcium oxalate monohydrate (COM) and the inside of uric acid (UA). Proteomic analysis identified 37 and 14 proteins from the inside and outside of the stone, respectively, as matrix proteins. The proteins that were identified in an ethylenediaminetetraacetic acid (EDTA) fraction were able to bind calcium ions. Thus, calcium-binding proteins may play a significant role in the formation of urinary stones in patients with hyperuricemia.

Isolation and characterization of calmodulin from a molluscan smooth muscle

Calmodulin was purified from the anterior byssal retractor muscle (ABRM) of a mollusc Mytilus edulis. Ca2+-induced conformational changes in the ABRM calmodulin could be demonstrated by polyacrylamide gel electrophoresis, by u.v. absorption spectrum and by circular dichroic spectrum. The amino acid composition of the ABRM calmodulin closely resembled that of other invertebrate calmodulins. The ABRM calmodulin was less effective in activating rat brain phosphodiesterase than vertebrate calmodulins.

Detection of prothrombin and osteopontin in a renal stone found in a hyperuricemic patient using 2D-PAGE and LC-MS analysis.

The liquid chromatography‐mass spectrometry (LC‐MS) following on from the two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) technique was applied for the analysis of proteins in a renal stone found in a hyperuricemic patient. This technique was sensitive enough to detect small quantities of proteins even in a renal stone.

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.

Development of LC-MS Method for Detection of Mutant Uromodulin Protein

Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 → 381, 471, 567, and 679 for GWHWE and m/z 688 → 381, 445, 541, and 653 for GWHC*E) were detected.