Masashi Shimahara

Urinary biomarkers monitoring for experimental fluoride nephrotoxicity

Abstract An excess of sodium fluoride (135 mg F/kg body weight) was given in a single oral dose to male Wistar rats. Effects were investigated of fluoride-induced acute kidney intoxication on the time-dependent variations of urine volume. Also, of urinary fluoride ion (F−), α-glutathione-S-transferase (α-GST), N-acetyl-β-d-glucosaminidase (NAG), and creatinine (CR) concentrations. Fluoride administration strongly affects these urinary biochemical indices. Of the several biomarkers studied, α-GST is particularly useful as marker of S3 proximal tubule damage. We found that α-GST shows the strongest and more durable changes as a result of the large dose of F− given to the experimental animals. Our results suggest that the toxic effect of F− on the kidney may be more pronounced in the proximal tubule than the glomeruli region, and that the disorder of the proximal tubule is more serious in the S3 segment than S1 or S2 segment. α-GST proved to be a useful marker for the early detection and long-term observation of proximal renal tubular injury resulting from F− intoxication. The animal model should help to establish guidelines for the treatment of industrial workers suffering from acute renal failure resulting from accidental exposure to fluoride.

Determining whether a parotid tumor is in the superficial or deep lobe using magnetic resonance imaging

PURPOSE This study examined the usefulness of magnetic resonance imaging (MRI) in assessing whether parotid tumors were located in the superficial or deep lobe. PATIENTS AND METHODS Eight patients with parotid gland tumors underwent MRI using a spin echo pulse sequence. T1- and T2-weighted images were obtained. To assess tumor localization in the parotid gland, two anatomic landmarks were used: 1) a line connecting the lateral surface of posterior belly of digastric muscle and lateral surface of the cortical bone of ascending ramus (facial nerve [FN] line) and 2) the relationship to the retromandibular vein (RV). Results of all MRI examinations were compared with the surgical findings. RESULTS Seven of eight cases were correctly diagnosed using the FN line criterion. Five of eight cases were correctly diagnosed using the RV criterion. CONCLUSION MRI is an excellent modality to show tumor localization in the parotid gland.

Plasma biomarker discovery and validation for colorectal cancer by quantitative shotgun mass spectrometry and protein microarray

The development of a new plasma biomarker for early detection would be necessary to improve the overall outcome of colorectal cancer. Here we report the identification and validation of the ninth component of complement (C9) as a novel plasma biomarker for colorectal cancer by cutting‐edge proteomic technologies. Plasma proteins were enzymatically digested into a large array of peptides, and the relative quantity of a total of 94 803 peptide peaks was compared between 31 colorectal cancer patients and 59 age/sex‐matched healthy controls using 2D image‐converted analysis of liquid chromatography and mass spectrometry. The selected biomarker candidates were validated in 345 subjects (115 colorectal cancer patients and 230 age/sex‐matched healthy controls) using high‐density reverse‐phase protein microarrays. Plasma levels of Apo AI and C9 in colorectal cancer patients significantly differed from healthy controls with P values of 7.94 × 10−4 and 1.43 × 10−12 (Student’s t‐test), respectively. In particular, C9 was elevated in patients with colorectal cancer, including those with stage‐I and ‐II diseases (P = 3.01 × 10−3 and P = 1.13 × 10−5, respectively). Although the significance of the present study must be validated in an independent clinical study, the increment of plasma C9 may be applicable to the early detection of colorectal cancer. (Cancer Sci 2011; 102: 630–638)

Prolyl 4-Hydroxylation of α-Fibrinogen: A NOVEL PROTEIN MODIFICATION REVEALED BY PLASMA PROTEOMICS*

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL),” is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of α-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated α-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated α-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated α-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.

Reduced Plasma Level of CXC Chemokine Ligand 7 in Patients with Pancreatic Cancer

Background: Early detection is essential to improve the outcome of patients with pancreatic cancer. A noninvasive and cost-effective diagnostic test using plasma/serum biomarkers would facilitate the detection of pancreatic cancer at the early stage. Methods: Using a novel combination of hollow fiber membrane–based low-molecular-weight protein enrichment and LC-MS-based quantitative shotgun proteomics, we compared the plasma proteome between 24 patients with pancreatic cancer and 21 healthy controls (training cohort). An identified biomarker candidate was then subjected to a large blinded independent validation (n = 237, validation cohort) using a high-density reverse-phase protein microarray. Results: Among a total of 53,009 MS peaks, we identified a peptide derived from CXC chemokine ligand 7 (CXCL7) that was significantly reduced in pancreatic cancer patients, showing an area under curve (AUC) value of 0.84 and a P value of 0.00005 (Mann–Whitney U test). Reduction of the CXCL7 protein was consistently observed in pancreatic cancer patients including those with stage I and II disease in the validation cohort (P < 0.0001). The plasma level of CXCL7 was independent from that of CA19-9 (Pearsons r = 0.289), and combination with CXCL7 significantly improved the AUC value of CA19-9 to 0.961 (P = 0.002). Conclusions: We identified a significant decrease of the plasma CXCL7 level in patients with pancreatic cancer, and combination of CA19-9 with CXCL7 improved the discriminatory power of the former for pancreatic cancer. Impact: The present findings may provide a new diagnostic option for pancreatic cancer and facilitate early detection of the disease. Cancer Epidemiol Biomarkers Prev; 20(1); 160–71. ©2011 AACR.

Identification of Adipophilin as a Potential Plasma Biomarker for Colorectal Cancer Using Label-Free Quantitative Mass Spectrometry and Protein Microarray

Background: The aim of this study was to identify a new plasma biomarker for use in early detection of colorectal cancer. Methods: Using the combination of hollow fiber membrane (HFM)-based low-molecular weight protein enrichment and two-dimensional image converted analysis of liquid chromatography and mass spectrometry (2DICAL), we compared the plasma proteome of 22 colorectal cancer patients with those of 21 healthy controls. An identified biomarker candidate was then validated in two larger cohorts [validation-1 (n = 210) and validation-2 (n = 113)] using a high-density reverse-phase protein microarray. Results: From a total of 53,009 mass peaks, we identified 103 with an area under curve (AUC) value of 0.80 or higher that could distinguish cancer patients from healthy controls. A peak that increased in colorectal cancer patients, with an AUC of 0.81 and P value of 0.0004 (Mann–Whitney U test), was identified as a product of the PLIN2 gene [also known as perilipin-2, adipose differentiation-related protein (ADRP), or adipophilin]. An increase in plasma adipophilin was consistently observed in colorectal cancer patients, including those with stage I or stage II disease (P < 0.0001, Welchs t test). Immunohistochemical analysis revealed that adipophilin is expressed primarily in the basal sides of colorectal cancer cells forming polarized tubular structures, and that it is absent from adjacent normal intestinal mucosae. Conclusions: Adipophilin is a plasma biomarker potentially useful for the detection of early-stage colorectal cancer. Impact: The combination of HFM and 2DICAL enables the comprehensive analysis of plasma proteins and is ideal for use in all biomarker discovery studies. Cancer Epidemiol Biomarkers Prev; 20(10); 2195–203. ©2011 AACR.

Epidemiological study of malignant tumors in the oral and maxillofacial region: survey of member institutions of the Japanese Society of Oral and Maxillofacial Surgeons, 2002

We studied 1809 patients with oral cancer who visited and were treated, in 2002, at the 148 institutions certified as training facilities by the Japanese Society of Oral and Maxillofacial Surgeons. Of these institutions, 39 are dental university hospitals, 44 are medical university hospitals, 64 are general hospitals, and for 1 institution, the classification was not known. The patients consisted of 1071 (59.2%) males and 738 (40.8%) females (male: female ratio, 1.45:1), who had a average age of 65.2 years. The tongue (40.2%) was the most common site affected, followed by the gingiva (32.7%), buccal mucosa (10.1%), and oral floor (9.0%). There were 6 cases of multiple intraoral cancers. On histopathological examinations, squamous cell carcinoma (88.7%) was the most common type found, followed by adenoid cystic carcinoma (2.1%), and mucoepidermoid carcinoma (1.7%). Cases classified as T2N0 were the most common (32.1%), followed by T1N0 (21.4%), T4N0 (8.0%), and T2N1 (7.6%). Distant metastasis occurred in 17 patients (1.0%). Nonepithelial tumors, among which malignant melanoma was the most common type, accounted for 1.8% of the tumors. The sizes of the nonepithelial malignant tumors ranged from 1.0 to 7.0 cm, with an average size of 3.7 cm.

Toxicokinetics of intravenous fluoride in rats with renal damage caused by high-dose fluoride exposure

Abstract Fluoride (F) complexes are used in some fields of industry and medicine. F excretion mainly depends on kidney function. Urinary F concentration is measured to monitor the health of workers exposed to F. The toxicokinetics of F were studied by analyzing plasma concentration of F after intravenous injection of 2.86, 5.71 and 8.57 mg/kg into male Wistar rats. A dose–response relationship was recognized between these F doses and renal tissue injury. Blood samples were removed at 0, 10, 20, and 30 min, and after 1, 2, 3, 4, 5, and 6 h after injection. Plasma concentration-vs-time profiles were evaluated by a nonlinear least-squares method for fitting data to polyexponential equations and calculation of relevant pharmacokinetic parameters. Results indicated that a two–compartment model could describe the elimination of F from plasma. The β rate constant, total plasma clearance (Cl) and first-order rate constants (K21, Kel) decreased, and the half-time of the β-phase (t1/2β) was significantly prolonged with increasing dose. The kidney is the main target organ for F toxicity. Acute exposure to high doses of F damages renal tissue and causes renal dysfunction. The Cl of F is mainly dependent on renal F excretion. Since severe kidney damage markedly affected the toxicokinetics of F and decreased its elimination, other nephrotoxic indicators and measurement of plasma F concentration are necessary for monitoring high-dose F exposure.

Glossodynia from Candida-Associated Lesions, Burning Mouth Syndrome, or Mixed Causes

OBJECTIVE Candida-associated lesions (CALs) and burning mouth syndrome (BMS) may induce glossodynia without objective manifestations. We investigated patients with glossodynia to examine the relationship between CAL and BMS. PATIENTS AND METHODS A visual analog scale was used to divide 95 patients with glossodynia into three groups according to intensity of pain at rest and when eating. Group A was the functional pain group; group B was the nonfunctional pain group; and group C was a mixed pain group. Antifungal treatment was scheduled for patients with suspected Candida infection by clinical, mycological, or cytological criteria. RESULTS Culture tests and direct examination results indicated that group A had high Candida positivity (73.0% by culture and 59.5% by direct examination), and showed a good response to antifungal treatment (75.7%). Antifungal treatment was not useful in group B. This was supported by a low Candida infection rate, as determined by direct examination (3.1%). For group C, Candida positivity and antifungal treatment effectiveness were between groups A and B. Furthermore, six patients in group C showed complete remission of functional pain by antifungal treatment only. Favorable outcomes were obtained for 23 patients (10 in group B and 13 in group C), who received antidepressant treatment. CONCLUSION These results suggested that glossodynia was Candida-associated in group A, and BMS-induced in group B, while group C contained patients with both CAL and BMS.

Usefulness of culture test and direct examination for the diagnosis of oral atrophic candidiasis

Background  Culture test and direct microscopy, which are currently used in the diagnosis of oral candidiasis, can yield false‐negative results.