Incisura tangent method to determine the transsyndesmotic axis for syndesmotic fixation.

BACKGROUND: Clamping along the transsyndesmotic (TS) axis decreases the risk of malreduction when reducing syndesmotic diastasis. We aimed to measure the difference between the TS axis and the axis determined by the newly proposed fluoroscopic incisura tangent (IT) method. The measurements were compared to those between the TS axis and those based on the center-center (CC) and talar dome lateral (TL) methods. METHODS: We analyzed computed tomographic images of 43 normal ankles. The IT view was simulated using a digitally reconstructed radiograph, in which the anterior and posterior fibular incisura tubercles overlapped on the internally rotated anteroposterior view. The interaxis angle between the TS and the axes determined by the IT method was measured on the axial computed tomographic image corresponding to the radiographic image. The same procedure was repeated using the CC and TL methods. The measured values were compared between the three methods using a one-factor analysis of variance. Furthermore, the measurements of the anteverted and retroverted incisurae were compared for each fluoroscopic method. RESULTS: The mean interaxis angles between the TS were - 0.5 degrees, 6.3 degrees, and - 1.8 degrees for the IT, CC, and TL methods, respectively, with a significantly larger value for the CC method than for the IT and TL methods (P < .001). No significant difference was found in the interaxis angle in the anteverted (-0.1 degrees) and retroverted (-1.0 degrees) incisurae when using the IT method (P = .15). The angles in the retroverted incisurae were larger than those of the anteverted incisurae for the CC and TL methods. CONCLUSION: The fluoroscopic IT method accurately estimated the TS axis. The interaxis angles were consistent, regardless of the incisura anatomy. The fluoroscopic method can be used to clamp and fix the syndesmosis along the TS axis. LEVEL OF EVIDENCE: Ⅳ.

Lateral meniscal injury without medial meniscal injury indicates the existence of the Segond fracture in ACL-deficient knees.

Background/Objective: The purpose of this study was to find factors indicating the occurrence of the Segond fracture, a specific type of anterolateral ligament injury. Methods: From January 2015 to December 2017, we retrospectively reviewed the medical records of patients diagnosed with acute anterior cruciate ligament (ACL) injury who underwent reconstruction within 90 days of injury. Diagnosis of the Segond fracture was determined either by magnetic resonance imaging or plain radiographs. Factors examined were: age at surgery, sex, body mass index (kg/m2), status of menisci, and activities led to ACL injury. After univariate screening, multivariate logistic regression analyses were performed. Patients were divided into four groups based on the presence of lateral meniscal (LM) and/or medial meniscal (MM) injuries and compared with respect to the occurrence of Segond fractures. Results: A total of 375 patients were included (163 males, 212 females), with mean age 25.8 years old. Among them, 22 of 375 (5.9 %) had a Segond fracture. We identified injured lateral menisci (adjusted odds ratio (aOR), 3.029; 95 % Confidence Interval (CI), 1.206-7.609; P = 0.018), intact medial menisci (aOR, 0.229; 95 % CI, 0.065-0.810; P = 0.022), and higher body mass index (aOR, 1.102; 95 % CI, 1.008-1.205; P = 0.034) as factors indicative of the occurrence of Segond fracture. LM injury without MM injury suggested the existence of a Segond fracture. Conclusion: LM injury without a MM injury indicated the occurrence of a Segond fracture. Higher body mass index also increased the risk for Segond fracture occurrence.

Impact of telephone follow-up on hepatocellular carcinoma patients receiving oral chemotherapy from an ambulatory-care setting.

INTRODUCTION: In recent years, most molecular target drugs have been administered orally, as prescribed at ambulatory services in hospitals and at patients' homes. Telephone follow-up is increasingly being used in clinical practice for patients needing additional support post-discharge and for the prevention of hospital readmissions. The purpose of this study was to clarify the clinical benefits of telephone follow-up while administering oral anticancer drugs. METHODS: This was a single-center, observational, retrospective study. We evaluated hepatocellular carcinoma patients who received sorafenib or lenvatinib between March 2010 and February 2018. The primary endpoint was the incidence of adverse events. RESULTS: From the total of 130 patients, 83 patients received telephone follow-up and 47 did not. The incidence of hand-foot skin reactions significantly reduced in patients with telephone follow-up (odds ratio (OR) 3.69, 95% confidence interval (CI) 1.16-11.8, p = 0.020). The median durations (ranges) of adherence to oral chemotherapy were 259 days (15-1730) for the telephone follow-up group and 121 days (14-1105) for the no-telephone follow-up group (p < 0.001). Moreover, the disease control rate was significantly higher in the telephone follow-up group (OR 2.52, 95% CI 1.15-5.53, p = 0.020). CONCLUSIONS: Remote interventions, such as telephone follow-up, are useful means of managing adverse events in patients receiving oral anticancer drugs and can lead to improved treatment results.

Internet search analysis on the treatment of rheumatoid arthritis: What do people ask and read online?

OBJECTIVES: This study aimed to characterize the content of frequently asked questions about the treatment of rheumatoid arthritis (RA) on the internet in Japan and to evaluate the quality of websites related to the questions. METHODS: We searched terms on the treatment of RA on Google and extracted frequently asked questions generated by the Google "people also ask" function. The website that answered each question was also obtained. We categorized the questions based on the content. The quality of the websites was evaluated using the brief DISCERN, Journal of American Medical Association benchmark criteria, and Clear Communication Index. RESULTS: Our search yielded 83 questions and the corresponding websites. The most frequently asked questions were regarding the timeline of treatment (n = 17, 23%) and those on the timeline of the clinical course (n = 13, 16%). The median score of brief DISCERN was 11 points, with only 7 (8%) websites having sufficient quality. Websites having sufficient quality based on the Journal of American Medical Association benchmark criteria and Clear Communication Index were absent. CONCLUSIONS: The questions were most frequently related to the timeline of treatment and clinical course. Physicians should provide such information to patients with RA in the counseling and education materials.

Validity of the Center-Center Method for the Syndesmotic Fixation Axis Compared to the Transsyndesmotic Axis.

BACKGROUND: Placement of clamp forceps along the transsyndesmotic (TS) axis reduces the risk of iatrogenic syndesmotic malreduction during ankle fracture surgery with tibiofibular diastasis. This study aimed to measure the difference between the TS axis and the axis determined by an intraoperative fluoroscopic technique using the center-center (CC) method. We also compared the values obtained when the CC method was performed at different heights from the tibial plafond. METHODS: We evaluated the computed tomography scans of 150 patients with normal syndesmosis. The CC method was simulated using digitally reconstructed radiographs. The TS and CC axes were projected onto an axial computed tomographic image linked to digitally reconstructed radiography. The angle between the two axes (interaxis angle) and the distance between the intersection of these axes and the medial tibial cortex (interaxis distance) were measured. The procedures were performed 0, 10, and 20 mm proximal to the tibial plafond, and the measurements were compared among the different heights using a 1-way repeated-measures analysis of variance. RESULTS: The TS axis was found to be externally rotated to the CC axis, with an interaxis angle of 8.5 degrees (SD, 6.8 degrees). The interaxis angle increased from 1.9 degrees at a height of 0 mm to a greater angle at higher heights (P < .001). The overall interaxis distance was 7.7 (SD, 6.3) mm, increasing from 2.0 mm at a height of 0 mm to a greater distance at higher heights (P < .001). CONCLUSION: The TS axis was externally rotated relative to the CC axis, and the difference between the 2 axes was greater when the CC method was performed on the higher heights from the tibial plafond. To clamp the syndesmosis along the TS axis, the CC method should be applied close to the ankle joint. LEVEL OF EVIDENCE: Level IV, case series.

Readability and quality of online patient resources regarding knee osteoarthritis and lumbar spinal stenosis in Japan.

BACKGROUND: This study aimed to quantify the readability and quality of online patient resources on knee osteoarthritis and lumbar spinal stenosis in Japan. METHODS: Three search engines (Google, Yahoo, and Bing) were searched for the terms knee osteoarthritis and lumbar spinal stenosis. The first 30 websites of each search were screened. Duplicate websites and those unrelated to the searched diseases were excluded. The remaining 125 websites (62 on knee osteoarthritis, 63 on lumbar spinal stenosis) were analyzed. The text readability was assessed using two web-based programs (Obi-3 and Readability Research Lab) and lexical density. Website quality was evaluated using the DISCERN score, Clear Communication Index, and Journal of American Medical Association benchmark criteria. RESULTS: Readability scores were high, indicating that the texts were difficult to understand. Only 24 (19%) and six (5%) websites were classified as average difficulty readability according to Obi-3 and Readability Research Lab, respectively. The overall quality of information was low, with only four (3%) being rated as having sufficient quality based on the Clear Communication Index and Journal of American Medical Association benchmark criteria. None of the websites satisfied the DISCERN quality criteria. CONCLUSIONS: Patient information on Japanese websites regarding knee osteoarthritis and lumbar spinal stenosis were difficult to understand. Moreover, the quality of the websites was insufficient. Orthopaedic surgeons should contribute to the creation of high-quality easy-to-read websites to facilitate patient-physician communication.

Peroneus brevis tear caused by an impingement between hypertrophied peroneal tubercle and lateral malleolus.

We report a 15-year-old female patient who sustained peroneus brevis injury caused by an impingement between the hypertrophied peroneal tubercle and lateral malleolus. The patient had pain for 3 years in the lateral side of her left ankle with unsuccessful conservative treatment. The oblique sagittal images of 3-dimensional magnetic resonance imaging and ultrasonography were useful in depicting the peroneus brevis injury and identifying the location of impingement between the hypertrophied peroneal tubercle and the tip of the lateral malleolus. The flatfoot deformity of the patient further aggravated the impingement. The patient was treated surgically, with excision of the enlarged tubercle and tendon repair. The ankle pain resolved 12 months postoperatively. Although rare, clinicians should recognize this condition as the cause of lateral ankle pain.

Efficacy and Safety of Esaxerenone in Hypertensive Patients with Diabetic Kidney Disease: A Multicenter, Open-Label, Prospective Study.

INTRODUCTION: Clinical data of esaxerenone in hypertensive patients with diabetic kidney disease (DKD) are lacking. We evaluated the efficacy and safety of esaxerenone in patients with DKD and an inadequate response to blood pressure (BP)-lowering treatment. METHODS: In this multicenter, open-label, prospective study, patients were divided into urinary albumin-to-creatinine ratio subcohorts (UACR < 30, 30 to < 300, and 300 to < 1000 mg/gCr). Esaxerenone was initiated at 1.25 mg/day and followed by incremental dose escalation based on BP and serum potassium level monitoring. The treatment period was 12 weeks. The primary endpoint was change in morning home systolic BP/diastolic BP (SBP/DBP) from baseline to end of treatment (EOT). Secondary endpoints included achievement rate of target BP, change in UACR from baseline, and safety. RESULTS: In total, 113 patients were enrolled. Morning home SBP/DBP significantly decreased from baseline to EOT in the total population (- 11.6/- 5.2 mmHg, both p < 0.001) and in all UACR subcohorts (all p < 0.001). The target BP achievement rate was 38.5%. Significant reductions in bedtime home and office BPs were also shown in the total population and all UACR subcohorts. UACR significantly improved from baseline to EOT in the total (- 50.9%, p < 0.001) and all UACR subcohorts (all p < 0.001). Incidence of serum potassium elevation as drug-related treatment emergent adverse events was 2.7%. The change from baseline in estimated glomerular filtration rate (eGFR) was - 4.8 mL/min/1.73 m2. CONCLUSION: Esaxerenone demonstrated a BP-lowering effect and improved albuminuria. The effects were consistent regardless of the severity of albuminuria without clinically relevant serum potassium elevation and eGFR reduction. CLINICAL TRIAL REGISTRATION: jRCTs06119002.

The Association of Postprandial Triglyceride Variability with Renal Dysfunction and Microalbuminuria in Patients with Type 2 Diabetic Mellitus: A Retrospective and Observational Study.

OBJECTIVE: We examined whether or not day-to-day variations in lipid profiles, especially triglyceride (TG) variability, were associated with the exacerbation of diabetic kidney disease. METHODS: We conducted a retrospective and observational study. First, 527 patients with type 2 diabetes mellitus (DM) who had had their estimated glomerular filtration rate (eGFR) checked every 6 months since 2012 for over 5 years were registered. Variability in postprandial TG was determined using the standard deviation (SD), SD adjusted (Adj-SD) for the number of measurements, and maximum minus minimum difference (MMD) during the first three years of follow-up. The endpoint was a ≥40% decline from baseline in the eGFR, initiation of dialysis or death. Next, 181 patients who had no micro- or macroalbuminuria in February 2013 were selected from among the 527 patients for an analysis. The endpoint was the incidence of microalbuminuria, initiation of dialysis, or death. RESULTS: Among the 527 participants, 110 reached a ≥40% decline from baseline in the eGFR or death. The renal survival was lower in the higher-SD, higher-Adj-SD, and higher-MMD groups than in the lower-SD, lower-Adj-SD, and lower-MMD groups, respectively (log-rank test p = 0.0073, 0.0059, and 0.0195, respectively). A lower SD, lower Adj-SD, and lower MMD were significantly associated with the renal survival in the adjusted model (hazard ratio, 1.62, 1.66, 1.59; 95% confidence intervals, 1.05-2.53, 1.08-2.58, 1.04-2.47, respectively). Next, among 181 participants, 108 developed microalbuminuria or death. The nonincidence of microalbuminuria was lower in the higher-SD, higher-Adj-SD, and higher-MMD groups than in the lower-SD, lower-Adj-SD, and lower-MMD groups, respectively (log-rank test p = 0.0241, 0.0352, and 0.0474, respectively). CONCLUSIONS: Postprandial TG variability is a novel risk factor for eGFR decline and the incidence of microalbuminuria in patients with type 2 DM.

Which Is a Better Skeletal Muscle Mass Index for the Evaluation of Physical Abilities: The Present Height or Maximum Height?

Objective Sarcopenia and osteoporosis often coexist in older adults. Sarcopenia is diagnosed using the skeletal muscle mass index (SMI), which is calculated as the appendicular skeletal muscle mass (ASM)/(present height)2, although patients with osteoporosis frequently have a loss of body height. We therefore investigated whether the present height or maximum height is more useful for calculating the SMI in the evaluation of physical abilities. Methods We conducted a cross-sectional study to investigate the association of the SMI with physical abilities, such as the grip strength and gait speed, in 587 postmenopausal women. The SMI was evaluated using whole-body dual-energy X-ray absorptiometry (DXA). The SMI [(ASM)/(present height)2], modified SMI (mSMI) [(ASM)/(maximum height)2], and SMI difference (ΔSMI) (mSMI-SMI) were calculated. Results Age and body mass index (BMI)-adjusted regression analyses showed that the SMI (ß=0.30, p<0.001 and ß=0.14, p=0.034) and mSMI (ß=0.40, p<0.001 and ß=0.29, p<0.001) were positively associated while the ΔSMI was negatively associated with the grip strength and gait speed (ß=-0.15, p<0.001 and ß=-0.24, p<0.001, respectively). Furthermore, the age, BMI, and presence of osteoporotic fractures-adjusted logistic regression analyses showed that a low mSMI (<5.4 kg/m2) was significantly associated with a low grip strength (<18 kg) and slow gait speed (1.0 m/s) [odds ratio (OR) =2.45, 95% confidence interval (CI) =1.52-3.95 per SD increase, p<0.001; and OR=1.73, 95% CI=1.01-2.96, p=0.042, respectively], although a low SMI showed no such relationship (p=0.052 and p=0.813, respectively). Conclusion The mSMI using the maximum height is more useful for evaluating physical abilities than conventional SMI estimation in postmenopausal women.

Optimum Conditions for the Efficacy and Safety of Cryofiltration Apheresis: An Analysis of Circuit Temperatures Depending on Plasma Flow Rate and Cooling Coil Lengths/Turns.

A system providing both appropriate cooling and warming are needed for the efficacy and safety of cryofiltration (CF) plasmapheresis. We measured some points of CF circuit temperatures with varying plasma flow rates (QP = 10-40 mL/min) and the numbers of connecting cooling coils (one or two) under the conditions of blood flow rate (QB ) 100 mL/min with 7700-mm coil length, 19 turns, and 50-mL priming volume. We measured the respective temperatures of each point of starting/returning for an extracorporeal circuit (TA /TV ), intracooling coil (TC ), and post-plasma fractionator (PF) (TPF ). The subtraction of TV from TA (ΔT) was used as an indicator of safe return. There were no significant differences in TC , TPF , or ΔT in accordance with each QP between that of one and two coils. All of the Tc values under the condition QP ≤ 20 mL/min achieved <4°C. The TPF under the condition QP ≥ 20 mL/min was not significantly different compared to that of QP 30 mL/min (the lowest condition). Although the ΔT increased depending on the QP increase, the ΔT under the condition QP ≤ 15 mL/min was not significantly different from that of the control (one-way double-filtration plasmapheresis [DFPP]) group. We conclude that (i) one coil is enough for effective cooling in CF, and (ii) an ideal QP that fulfills the required conditions for both effective cooling and sufficient warming of returning fluid does not exist, but QP from 15 to 20 mL/min may be a relevant range.

Analysis of protein removal properties during cryofiltration apheresis using the Evaflux-5A plasma fractionator in a patient with hepatitis C virus-associated cryoglobulinemic glomerulonephritis.

Cryofiltration (CF) is a technique in which separated plasma is chilled before being subjected to a plasma fractionator (PF), leading to cryoglobulin precipitation or cryogel formation. In CF using the Evaflux-5A as the PF, there is no consensus on the necessity of albumin supplementation, and when or how often the PF column should be washed. We analyzed the sieving effects of various solutes (albumin, IgG, IgM, LDL, HCV-RNA, and cryoglobulin) depending on transmembrane pressure (TMPPF ) during CF using the Evaflux-5A in a patient with hepatitis C virus-associated cryoglobulinemic glomerulonephritis. Five CF treatments were initially performed and a sixth one later, at disease recurrence. Quantitative detection of cryoglobulin and a marked rise in TMPPF to 400 mm Hg were observed only at the first and sixth treatment, and albumin losses during these treatments were very high, at 16.8 g, and 14.6 g, respectively, while those of others (from the second to fifth) were 6.7 g, 6.4 g, 5.9 g, and 7.0 g, respectively. The sieving coefficients (SCs) of both albumin and IgG were stable (0.8-1.0) at TMPPF < 200 mm Hg, but significantly decreased at TMPPF ≥ 200 mm Hg (P < 0.01). The SC of IgM tended to decrease at TMPPF ≥ 200 mm Hg, but not significantly, while that of LDL was zero regardless of the TMPPF . Albumin loss per treatment likely depends on degree of TMPPF rise, which is mainly affected by the patient's cryoglobulinemic status. In CF using Evaflux-5A, washing the PF column to keep TMPPF < 200 mm Hg during treatment may be a recommended for selective removal and albumin salvage.

[Verification of reduction in preparation time and cost of cyclophosphamide when using the closed-system drug transfer device].

The closed-system transfer device (CSTD), which is used to prevent the exposure of medical staff to anticancer drugs, has been reported to allow safe preparation and administration of these drugs to patients. At many medical institutions, however, the use of such devices is limited to select anticancer drugs. This could be attributable to the longer preparation time compared to the conventional injection technique with a syringe and needle, as well as the fact that the anticancer drugs are too expensive to be covered by the remuneration available for medical services. Against this background, we measured the time required to prepare cyclophosphamide(CPA)and estimated the cost incurred. Our results indicated that the preparation time for either a single dose of 100 mg CPA or a combination of 100 mg CPA and 500 mg of another drug(100mg+500 mg group)was significantly longer than that for 500 mg of a single drug. On the other hand, use of a CSTD reduced the total cost (drug cost+CSTD cost)on switching to a single dose of 500 mg, resulting in a 5-year savings as follows: 3,755,217 yen for ChemoCLAVE®, 6,302,622 yen for PhaSeal®, and 2,698,451 yen for Chemosafe®. These findings suggest that the appropriate selection of drugs, including a large standard dose of CPA, allows shortened preparation time and reduced total drug cost as well as CSTD cost.

Clival chordoma manifesting as nasal bleeding. A case report.

Chordoma is a rare cartilaginous tumor, for which bleeding presentation is unusual. We report a case of rare hemorrhaged clival chordoma, which was diagnosed correctly by magnetic resonance imaging. A 32-year-old man presented with nasal bleeding. The tumor was totally removed via a trans-sphenoidal approach, from which the surgical specimen confirmed chordoma. Epistaxis seemed to be caused by the spreading of the intratumoral hemorrhage into the sphenoid sinus. This case demonstrates the importance of an exact differential diagnostic evaluation, including chordoma, by use of modern imaging techniques for nasal bleeding.

Cloning of a G-protein-coupled receptor that shows an activity to transform NIH3T3 cells and is expressed in gastric cancer cells.

The present study was directed towards the identification of novel factors involved in the transformation process leading to the formation of gastric cancer. A cDNA library from human gastric cancer cells was constructed using a retroviral vector. Functional cloning was performed by screening for transformation activity in transduced NIH3T3 cells. Six cDNA clones were isolated, including one encoding the elongation factor 1alpha subunit, which was already known to play a role in tumorigenesis. One cDNA (clone 56.2), which was repeatedly isolated during the course of screening, encoded a protein identical to a G-protein-coupled receptor protein, GPR35. In addition, another cDNA clone (72.3) was found to be an alternatively spliced product of the GPR35 gene, whereby 31 amino acids were added to the N-terminus of GPR35. Hence, the proteins encoded by clones 56.2 and 72.3 were designated GPR35a and GPR35b, respectively. RT-PCR experiments revealed that GPR35 gene expression is low or absent in surrounding non-cancerous regions, while both mRNAs were present in all of the gastric cancers examined. The level of 72.3-encoded mRNA was consistently significantly higher than that of 56.2 encoded mRNA. An expression pattern similar to that observed in gastric cancers was detected in normal intestinal mucosa. Based on the apparent transformation activities of the two GPR35 clones in NIH3T3 cells, and the marked up-regulation of their expression levels in cancer tissues, it is speculated that these two novel isoforms of GPR35 are involved in the course of gastric cancer formation.

Anti-viral actions and viral dynamics in the early phase of three different regimens of interferon treatment for chronic hepatitis C: differences between the twice-daily administration of interferon-beta treatment and the combination therapy with interferon-alpha plus ribavirin.

To improve the efficacy of interferon (IFN) treatment for chronic hepatitis C, we have proposed the twice-daily administration of IFN-beta as a promising induction therapy. In this study, we demonstrated differences between the clearance of circulating HCV-RNA and the induction of anti-viral actions during the first 2 weeks of treatment. Nine patients with a high viral load and genotype 1b were randomly assigned to 3 groups: group A received 3MU of IFN-beta twice a day at intervals of 5 and 19 h; group B received 3MU of IFN-beta twice a day at intervals of 10 and 14 h; group C received 6MU of IFN-alpha once a day with ribavirin. The expression of OAS2, PKR, and MxA in peripheral blood mononuclear cells (PBMCs) were quantified by real-time polymerase chain reaction method. The viral clearance showed a bi-phasic pattern, and those in the second phase of groups A and B were significantly steeper than that of group C. The peak level of OAS2 during the first phase was correlated with the first phase decay. The MxA expression tended to be higher in group A and B than in group C. The expression of these 3 proteins tended to decrease at day 6 in group C, but increase in groups A and B. These might make differences in the viral decay during the second phase

Hepatitis B virus gene in liver tissue promotes hepatocellular carcinoma development in chronic hepatitis C patients.

The hepatitis B virus (HBV) gene has been detected in hepatocellular carcinoma (HCC) tissue negative for the hepatitis B surface antigen and positive for the hepatitis C virus (HCV) antibody, but the precise role of the HBV gene in hepatocarcinogenesis has yet to be clarified. We studied the HBV gene in liver tissue several years before the emergence of HCC. Eleven patients diagnosed with HCV-positive chronic liver disease and who developed HCC were assigned to group A. HBV DNA was detected in 8 of the 11 patients (73%). Twenty-five patients, who did not develop HCC, were selected as group B. Six of the group B patients were classified as DNA-positive (24%). The HBV DNA in liver tissue was found to be significantly related to HCC development (P < 0.01). Thus, the presence of the HBV gene in patients with chronic HCV associated-liver injury appears to promote hepatocarcinogenesis, although prospective studies are needed to confirm this result.

Accurate measurement of near-micromolar oxygen concentrations in aqueous solutions based on enzymatic extradiol cleavage of 4-chlorocatechol: applications to improved low-oxygen experimental systems and quantitative assessment of back diffusion of oxygen from the atmosphere.

An enzymatic method for measuring the O(2) concentrations of aqueous solutions was developed by involving 4-chlorocatechol and catechol 2,3-dioxygenase from Pseudomonas putida. With this system, the amount of O(2) in a sample solution can be measured as the amount of 5-chloro-2-hydroxymuconate semialdehyde formed through the enzyme reaction. The product was stable and its anion exhibited strong absorption around 380 nm (molar absorption coefficient of 4.3 x 10(4) M(-1) cm(-1), pK value of 5.4). A sensitive HPLC method involving a BioAssist Q column was developed to individually quantify the products derived from 4-chlorocatechol and catechol. When the O(2) concentration in a sample solution sealed in a vial was lowered from the air-saturation level by means of the amount enzymatically reacted with a known amount of catechol, the concentration of remaining O(2) could be successfully measured by the HPLC method. We developed devices through which reagents could be added to solutions sealed in cuvettes or the vessel of an oxygen electrode system under a flow of argon. By applying these devices, the submicromolar O(2) concentration of an anoxic solution and the back diffusion of O(2) from the atmosphere could be directly determined for the first time. The K(m) values of the dioxygenase and an ascorbate oxidase for oxygen were also determined to be 7.2 (at pH 7.5) and 114 microM (at pH 6.5), respectively, at 25 degrees C.