Detection of homogentisic acid by electrospray ionization mass spectrometry.

OBJECTIVE: Homogentisic acid (HGA) is excreted in excessive amounts in the urine of patients with alkaptonuria, which is a hereditary metabolic disorder of phenylalanine and tyrosine. Therefore, the detection of HGA in urine is useful for the diagnosis of alkaptonuria. To evaluate the detection of HGA, we confirmed the color shift of HGA solutions and analyzed them by electrospray ionization mass spectrometry (ESI-MS). METHODS: We observed the color change of the HGA solutions under different pH conditions (pH 6.0, 7.0, and 8.0) and examined the influences of adding potassium hydroxide (KOH) and ascorbic acid (AA) to the HGA solutions. Then, we analyzed the chemical reaction in HGA solutions using ESI-MS. RESULTS: The HGA solution at pH 8.0 became brown after incubation at room temperature for 24 h and became darker brown with the addition of KOH; however, HGA solutions at pH 6.0 and 7.0 showed no color changes. The brown color change of the HGA solution at pH 8.0 was also inhibited by AA. Moreover, all HGA sample solutions showed the deprotonated molecular ion peak at m/z 167.035 in the negative ion mode after incubation at room temperature for 24 h and with the addition of KOH and AA. CONCLUSION: We identified the molecular ion of HGA in all sample solutions by ESI-MS, regardless of different pH conditions, color changes, or the presence of AA. These results suggest that spectral analysis by ESI-MS is suitable for the detection of HGA and the diagnosis of alkaptonuria.

Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

Presepsin (P-SEP) is a specific biomarker for sepsis. Monocytes produce P-SEP by phagocytosing neutrophil extracellular traps (NETs). Herein, we investigated whether M1 macrophages (M1 MΦs) are the primary producers of P-SEP after NET phagocytosis. We co-cultured M1 MΦs and NETs from healthy participants, measured P-SEP levels in the culture medium supernatant, and detected P-SEP using western blotting. When NETs were co-cultured with M1 MΦs, the P-SEP level of the culture supernatant was high. Notably, we demonstrated, for the first time, the intracellular kinetics of P-SEP production by M1 MΦs via NET phagocytosis: M1 MΦs produced P-SEP intracellularly 15 min after NET phagocytosis and then released it extracellularly. In a sepsis mouse model, the blood NET ratio and P-SEP levels, detected using ELISA, were significantly increased (p < 0.0001). Intracellular P-SEP analysis via flow cytometry demonstrated that lung, liver, and kidney MΦs produced large amounts of P-SEP. Therefore, we identified these organs as the origin of M1 MΦs that produce P-SEP during sepsis. Our data indicate that the P-SEP level reflects the trend of NETs, suggesting that monitoring P-SEP can be used to both assess NET-induced organ damage in the lungs, liver, and kidneys during sepsis and determine treatment efficacy.

Diagnostic Immunocytostaining with Peroxisome Proliferator-Activated Receptor-Gamma in Urine Cytology Samples.

INTRODUCTION: Urine cytology is a common method for detection of urothelial carcinoma (UC), however, is not high sensitivity. Improvement of the accuracy of cytodiagnosis using immunocytostaining as an auxiliary method is needed. This study aimed to determine the cytodiagnostic usefulness of peroxisome proliferator-activated receptor-gamma (PPAR-γ) immunocytostaining in urine cytology for the detection of UCs, particularly low-grade urothelial carcinomas (LGUC). METHODS: PPAR-γ immunocytostaining was performed for 37 UC cases and 26 benign cases. Among the UC cases, 22 cases were of the papillary proliferation type, not including the mixed type comprising both papillary and flat growth. Fifteen LGUC cases of all papillary proliferation types were included. For comparison, the same samples were also immunocytostained for p53 and Ki-67. RESULTS: Of the UC cases, 25 of 37 were positive for PPAR-γ, while 24 of the 26 benign cases were PPAR-γ-negative. Regardless of histological grading, 13 of the 22 UC cases with papillary proliferation were PPAR-γ-positive. In particular, PPAR-γ immunocytostaining showed higher sensitivity for LGUC cases than that of the other biomarkers. Regarding LGUC specifically, 4 of 10 cases not identified by primary cytology were detected by PPAR-γ immunocytostaining. CONCLUSION: PPAR-γ immunocytostaining enhances the accuracy of urine cytodiagnosis. Furthermore, PPAR-γ is a more useful immunobiomarker in urine cytology than p53 and Ki-67, the commonly used immunobiomarkers for malignant cell detection.

A spectrophotometric method for the determination of tryptophan following oxidation by the addition of sodium hypochlorite pentahydrate.

Tryptophan (Trp) is an essential amino acid that functions in various biological processes and human daily health. As the significant functions of Trp become more apparent, its measurement is becoming increasingly important in various situations. Herein, we improved the Trp color reaction based on the Hopkins-Cole reaction and established a simple colorimetric method for Trp determination using several different reagents, including sodium hypochlorite pentahydrate and monosodium glutamate. The detection method can be performed using safe materials, rather than conventional toxic substances, and induces a crimson color change with an absorption peak at 525 nm, enabling the quantification of Trp by simple spectrophotometry in just 10 min. This assay exhibited a linear detection range from 10 to 100 mg/L (R2 = 0.9996). The average recoveries in the spiked cerebrospinal fluid ranged from 90.5% to 104.3%, with a relative standard deviation of 0.27% (n = 3, 29.40 mg/L Trp) to 1.19% (n = 3, 72.90 mg/L Trp). This novel spectrophotometric method may enable many researchers and laboratory technicians to detect Trp in various sample solutions without expensive analytical instruments or complicated operations.

Overexpression of the PPAR-γ protein in primary Ta/T1 non-muscle-invasive urothelial carcinoma.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a well-known nuclear receptor that is activated in the nucleus to regulate several transcription factors. Its expression patterns have been examined in various types of cancer. The present study investigated the expression patterns of PPAR-γ in non-muscle-invasive urothelial carcinoma. The expression rates of PPAR-γ, p53 and Ki-67 were compared to determine whether PPAR-γ may be considered as an immunobiomarker for bladder cancer. The intensity and extent of PPAR-γ expression were evaluated in 79 cases of non-muscle-invasive urothelial carcinoma (30 cases of papillary carcinoma low-grade, 30 cases of high-grade and 19 cases of carcinoma in situ) and 30 non-malignant cases. The nuclear overexpression of PPAR-γ was frequently observed in non-muscle-invasive urothelial carcinoma (63/79 cases) but was rarely detected in non-malignant cases (2/30 cases). The histological proliferation types of non-muscle-invasive urothelial carcinoma revealed that PPAR-γ was more frequently overexpressed in papillary carcinoma (54/60 cases) than in carcinoma in situ (9/19 cases). Immunohistochemical staining demonstrated that PPAR-γ was more useful as an immunobiomarker than p53 or Ki-67 (diagnostic odds ratios; 55.13, 16.82 and 11.13, respectively). In summary, this study demonstrated that the expression patterns of PPAR-γ were associated with histological proliferation type and that PPAR-γ was expressed in the nuclei of papillary carcinoma cells. These findings suggested that immunohistochemical staining for PPAR-γ may be used to comprehensively detect non-muscle-invasive urothelial carcinoma.

Peroxisome proliferator-activated receptor-α expression is associated with histological type in human gastric carcinoma.

Gastric carcinoma is one of the most common types of cancer worldwide and a leading cause of cancer-related mortality. Gastric carcinoma is histologically subdivided into differentiated and undifferentiated carcinoma, with the latter including poorly differentiated carcinoma and signet ring cell carcinoma (SRCC). Poorly differentiated carcinoma and SRCC have a worse prognosis compared with differentiated carcinoma. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors and the PPAR-α subtype regulates important cellular functions, including cell proliferation, energy metabolism, oxidative stress, immune responses and cell differentiation. The aim of the present study was to elucidate the associations between clinicopathological factors and PPAR-α expression in patients with gastric carcinoma. The immunohistochemical staining of specimens obtained from 57 patients showed that PPAR-α expression was slightly weaker in undifferentiated carcinoma than in differentiated carcinoma (P<0.01). PPAR-α expression also significantly differed between poorly differentiated carcinoma (both positive and negative: 14/20, 70%) and SRCC (not expressed: 0/7, 0%) (P<0.01). However, PPAR-α expression was not significantly affected by age, lymph node invasion, venous invasion, lymph node metastasis, depth of invasion or stage. Collectively, the present results demonstrated that the downregulated expression of PPAR-α may play a key role in the biological transformation of tumors. Therefore, PPAR-α appears to be an important protein related to histology and may hold promise as a prognostic marker. Further studies with a larger number of subjects are needed to elucidate the relationship between PPAR-α expression and tumor progression and to analyze long-term clinical survival.

Novel absorbance peak of gentisic acid following the oxidation reaction.

Gentisic acid (GA), a metabolite of acetylsalicylic acid (ASA), and homogentisic acid (HGA), which is excreted at high levels in alkaptonuria, are divalent phenolic acids with very similar structures. Urine containing HGA is dark brown in color due to its oxidation. We recently reported a new oxidation method of HGA involving the addition of sodium hydroxide (NaOH) with sodium hypochlorite pentahydrate (NaOCl·5H2O), which is a strong oxidant. In the present study, we attempted to oxidize GA, which has a similar structure to HGA, using our method. We herein observed color changes in GA solution and analyzed the absorption spectra of GA after the addition of NaOH with NaOCl·5H2O. We also examined the oxidation reaction of GA using a liquid chromatography time-of-flight mass spectrometer (LC/TOF-MS). The results obtained indicated that GA solution had a unique absorption spectrum with a peak at approximately 500 nm through an oxidation reaction following the addition of NaOH with NaOCl·5H2O. This spectrophotometric method enables GA to be detected in sample solutions without expensive analytical instruments or a complex method.

Activation and Expression of Peroxisome Proliferator-Activated Receptor Alpha Are Associated with Tumorigenesis in Colorectal Carcinoma.

Peroxisome proliferator-activated receptor alpha (PPAR-α) belongs to the PPAR family and plays a critical role in inhibiting cell proliferation and tumorigenesis in various tumors. However, the role of PPAR-α in colorectal tumorigenesis is unclear. In the present study, we found that fenofibrate, a PPAR-α agonist, significantly inhibited cell proliferation and induced apoptosis in colorectal carcinoma cells. In addition, PPAR-α was expressed in the nucleus of colorectal carcinoma cells, and the expression of nuclear PPAR-α increased in colorectal carcinoma tissue compared with that of normal epithelium tissue (P<0.01). The correlation between the expression of nuclear PPAR-α and clinicopathological factors was evaluated in human colorectal carcinoma tissues, and the nuclear expression of PPAR-α was significantly higher in well-to-moderately differentiated adenocarcinoma than in mucinous adenocarcinoma (P<0.05). These findings indicate that activation of PPAR-α may be involved in anticancer effects in colorectal carcinomas, and nuclear expression of PPAR-α may be a therapeutic target for colorectal adenocarcinoma treatment.

Absorbance measurements of oxidation of homogentisic acid accelerated by the addition of alkaline solution with sodium hypochlorite pentahydrate.

The urine of patients with alkaptonuria turns dark brown due to the oxidation of homogentisic acid (HGA) to benzoquinone acetic acid (BQA), and this is accelerated by the addition of alkali. We recently reported that alkaptonuric urine and HGA after the addition of alkali showed characteristic peaks at 406 and 430 nm. In order to improve the sensitivity of our spectrometric method for the detection of HGA, we accelerated the oxidation of HGA to BQA using sodium hypochlorite pentahydrate (NaOCl·5H2O), which is a strong oxidant. In the present study, we measured the absorption spectra of alkaptonuric urine and HGA solution after the addition of sodium hydroxide (NaOH) or NaOH with NaOCl·5H2O and analyzed the oxidation reaction of HGA after alkalization using a liquid chromatography time-of-flight mass spectrometer (LC/TOF-MS) and nuclear magnetic resonance (NMR) spectrometry. We accelerated the oxidation of HGA to BQA by adding NaOH with NaOCl·5H2O, and this absorbance measurement was useful for more sensitively observing the oxidation of HGA than LC/TOF-MS and NMR spectroscopy. This quick and easy screening method may be suitable for the diagnosis of alkaptonuria.

Nuclear expression of claudin-3 in human colorectal adenocarcinoma cell lines and tissues.

Claudins are members of a large family of transmembrane proteins, which are essential for the formation of tight junctions and have a significant effect on the biological behavior of tumor progression. Previous studies have demonstrated that several claudins show aberrant expression patterns in numerous types of cancer. The present study investigated the expression and localization of claudin-3 and claudin-7 in human colorectal adenocarcinoma cell lines and tissues. The protein expression levels of claudin-3 and claudin-7 were determined using immunocytochemical and immunohistochemical staining. Claudin-3, but not claudin-7, exhibited nuclear localization in the human colorectal adenocarcinoma Caco-2 and SW620 cell lines. Surgically resected colorectal adenocarcinoma tissue specimens were obtained, and the associations between the expression of claudin-3 or claudin-7 and various clinicopathological parameters were analyzed. The membranous expression rates of claudin-3 and claudin-7 were 58.0 and 50.0%, while their nuclear expression rates were 22.0 and 2.0%, respectively. The membranous expression of claudin-3 and claudin-7 was not associated with any clinicopathological factors, whereas the nuclear expression of claudin-3 was associated with histological type and was significantly increased in colorectal mucinous adenocarcinomas compared with that in well- to moderately-differentiated colorectal adenocarcinomas (P<0.01). However, no associations were observed between the nuclear expression of claudin-7 and any clinicopathological parameter. In conclusion, the nuclear expression of claudin-3 in colorectal mucinous adenocarcinoma may be involved in the biological transformation of tumors. The results from the present study indicated that claudin-3 is an important protein associated with histological type and has potential as a prognostic marker. Although the mechanisms underlying the nuclear localization of claudin-3 in tumorigenesis have not yet been elucidated in detail, the present results indicated the potential of claudin-3 as a histopathological biomarker for colorectal adenocarcinomas.

Claudin-1, but not claudin-4, exhibits differential expression patterns between well- to moderately-differentiated and poorly-differentiated gastric adenocarcinoma.

Claudins are members of a large family of transmembrane proteins, which are essential in the formation of tight junctions and have previously been associated with the process of tumor progression. Studies have reported the aberrant expression of claudin-1 and claudin-4 in numerous types of cancer. The present study aimed to investigate the expression of claudin-1 and claudin-4 in gastric adenocarcinoma tissue. Surgically resected gastric adenocarcinoma tissue specimens were obtained from 94 patients. Protein expression levels of claudin-1 and claudin-4 were determined using immunohistochemical staining; the association between claudin-1 or claudin-4 expression and various clinicopathological parameters were then analyzed. In gastric adenocarcinoma specimens, the expression rates of claudin-1 and claudin-4 were 43.6 and 87.2%, respectively. Claudin-1 expression demonstrated a significant correlation with histological type (P<0.01) and was significantly higher in well- to moderately-differentiated gastric adenocarcinomas compared with poorly-differentiated tumors. However, no correlation was observed between claudin-4 expression in adenocarcinoma and clinicopathological parameters. In conclusion, downregulation of claudin-1 expression in poorly-differentiated gastric adenocarcinoma may be involved in the biological transformation of tumors. The present findings suggested that claudin-1 may be an important protein associated with histological type and therefore may have potential for use as a prognostic marker for gastric adenocarcinoma. Further studies are required to elucidate the precise mechanism of claudin expression and its involvement in tumor progression.