Effectiveness of Chemotherapy on Long-Term Survival in a Case of Advanced Juvenile Hepatocellular Carcinoma Without Viral Hepatitis Infection.

Hepatocellular carcinoma (HCC) usually occurs in settings of cirrhosis and chronic hepatitis B or C virus (HBV and HCV, respectively) infection; it is extremely rare in patients <40 years of age since viral- or alcohol-induced chronic hepatitis develops over a prolonged period. Juvenile HCC is mostly associated with persistent HBV infection; cases unrelated to HBV or HCV infection (non-B, non-C juvenile HCC) are sporadic and treated in the same way as classical HCC. A woman in her late 30s was diagnosed with HCC in a healthy liver; her imaging findings were typical of HCC with bone metastasis. She was administered a combination of tyrosine kinase inhibitors, immune checkpoint inhibitors, and vascular endothelial growth factor inhibitors. Throughout chemotherapy, the liver reserve was Grade A on the Child-Pugh classification and tumor markers remained under control without marked elevation. Our patient is the first reported long-term survivor of unresectable non-B, non-C juvenile HCC following chemotherapeutic treatment.

Alpha-fetoprotein-producing gastric cancer with ruptured liver metastasis mimicking hepatocellular carcinoma.

Alpha-fetoprotein (AFP)-producing gastric cancer is a rare subtype of gastric cancer known for its aggressive nature. We present an uncommon case of a 60s male with multiple liver tumors, initially suggested as hepatocellular carcinoma (HCC) by imaging. However, a subsequent gastric biopsy revealed a poorly differentiated adenocarcinoma with hepatoid features, and liver biopsy mirrored these findings. The disease progressed swiftly, with the patient representing owing to the spontaneous rupture of a metastatic liver tumor, an extremely rare occurrence, especially in metastatic liver cancers. Such ruptures in AFP-producing gastric cancer may be attributed to the tumor's rich blood flow. Distinctly differentiating this subtype from HCC is pivotal for apt management, as was evident in our case. The diagnosis was particularly challenging due to the similarities in imaging presentations between AFP-producing gastric cancer liver metastasis and HCC. This case underscores the need for vigilant diagnosis, emphasizing the importance of liver biopsy, especially in the absence of chronic liver disease. It also highlights the potential complications, like spontaneous rupture, associated with this rare form of gastric cancer.

Stem Cell Growth Factor-ß as a Predictive Biomarker of Response to Chemotherapy and Prognosis in Patients with Advanced-Stage Hepatocellular Carcinoma: A Retrospective Study.

In this retrospective study, we investigated the potential application of serum stem cell growth factor beta (SCGF-ß) as a biomarker for predicting the therapeutic response and prognosis in patients with hepatocellular carcinoma (HCC) undergoing atezolizumab and bevacizumab (Atz/Bev) combination therapy. Pre- and post-treatment serum SCGF-ß levels were measured and analyzed in relation to treatment outcomes and overall survival (OS). Pretreatment SCGF-ß levels were associated with treatment response. Patients with SCGF-ß levels exceeding the 163,295 pg/mL cutoff experienced significantly reduced OS, with a median OS of 12.03 months, compared to 28.87 months in those with SCGF-ß levels at or below this threshold. These findings suggest that SCGF-ß can serve as a predictive marker for clinical outcomes in HCC treatment, highlighting the need for prospective studies to further validate these results and clarify the mechanisms underlying SCGF-ß-related therapeutic resistance.

Successful Endoscopic Removal of Anisakis Larva that Deeply Invaded the Gastric Mucosa Using Jumbo Forceps.

Anisakiasis is a parasitic disease caused by the ingestion of raw or uncooked seafood infected with third-stage larvae of anisakid nematodes. Generally, the larvae parasites live at the surface of the mucosa, but in this case, the larva deeply invaded its head into the gastric mucosa and was not removable with conventional biopsy forceps. In our case, we demonstrated the usefulness of jumbo forceps to remove the Anisakis larva in such a situation.

New insights into the oxidation process from neutron and X-ray crystal structures of an O2-sensitive [NiFe]-hydrogenase.

[NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an O2-sensitive enzyme that is inactivated in the presence of O2 but the oxidized enzyme can recover its catalytic activity by reacting with H2 under anaerobic conditions. Here, we report the first neutron structure of [NiFe]-hydrogenase in its oxidized state, determined at a resolution of 2.20 Å. This resolution allowed us to reinvestigate the structure of the oxidized active site and to observe the positions of protons in several short hydrogen bonds. X-ray anomalous scattering data revealed that a part of the Ni ion is dissociated from the active site Ni-Fe complex and forms a new square-planar Ni complex, accompanied by rearrangement of the coordinated thiolate ligands. One of the thiolate Sγ atoms is oxidized to a sulfenate anion but remains attached to the Ni ion, which was evaluated by quantum chemical calculations. These results suggest that the square-planar complex can be generated by the attack of reactive oxygen species derived from O2, as distinct from one-electron oxidation leading to a conventional oxidized form of the Ni-Fe complex. Another major finding of this neutron structure analysis is that the Cys17S thiolate Sγ atom coordinating to the proximal Fe-S cluster forms an unusual hydrogen bond with the main-chain amide N atom of Gly19S with a distance of 3.25 Å, where the amide proton appears to be delocalized between the donor and acceptor atoms. This observation provides insight into the contribution of the coordinated thiolate ligands to the redox reaction of the Fe-S cluster.

Rapidly Progressing Sarcomatoid Hepatocellular Carcinoma after Needle Biopsy and Radiofrequency Ablation.

BACKGROUND Sarcomatoid hepatocellular carcinoma is a rare, primary malignant liver cancer. Its pathogenesis is unknown, but it often occurs in patients who have undergone repeated antitumor therapies for hepatocellular carcinoma. Sarcomatoid hepatocellular carcinoma is more likely to recur and has a worse prognosis than that of hepatocellular carcinoma. As no specific features have been identified in the symptoms, serological findings, or imaging findings, it is difficult to accurately diagnose the disease before surgical resection or autopsy. CASE REPORT An 83-year-old woman was diagnosed with hepatocellular carcinoma 20 years ago. Radiofrequency ablation was initially performed. Thereafter, invasive, non-surgical treatments were repeated. The most recent treatment was 4 years ago, during which computed tomography suggested recurrent hepatocellular carcinoma. However, upon needle biopsy, histological examination revealed spindle-shaped tumor cells and actively mitotic cells. Immunohistochemical analysis showed negative results for Arginase-1, HepPar1, and Glypican3 and positive results for AE1/AE3, CK7, and vimentin. Therefore, sarcomatoid hepatocellular carcinoma was diagnosed, which was treated with radiofrequency ablation but progressed rapidly thereafter. Considering the rapid disease progression, the patient was treated conservatively. However, the patient's general condition gradually deteriorated, resulting in death. CONCLUSIONS Compared with hepatocellular carcinoma, sarcomatoid hepatocellular carcinoma is more prone to recurrence and has a poorer prognosis. Therefore, aggressive surgical resection seems to be the most appropriate treatment for sarcomatoid hepatocellular carcinoma at present. Additional hepatic resection or follow-up imaging in a short period should be considered at the time of diagnosis of sarcomatoid hepatocellular carcinoma by biopsy, considering the risk of seeding or recurrence.

Prevalence of hepatitis C virus infection among men who have sex with men with human immunodeficiency virus-1 infection between 2010 and 2020 in Japan: A single-center retrospective cohort study.

INTRODUCTION: The high prevalence of hepatitis C virus (HCV) infection in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) is an important health issue. The purpose of this study is to investigate the actual prevalence of HCV infection among HIV-positive MSM in Japan. METHODS: This study is a single-center retrospective cohort study. We collected data of HIV-infected MSM who visited our hospital from January 2010 to December 2020, and evaluated HCV prevalence, course of HCV infection, and direct-acting antiviral (DAA) treatment efficacy in HIV-infected MSM. RESULTS: Overall, 1135 HIV-infected MSM had HCV antibody (Ab) tests during the observation period. The first anti-HCV Ab positive rate in HIV-infected MSM was 4% (45/1135), and the seroconversion rate of HCV antibody was 3.6% (39/1090). Treponema pallidum hemagglutination antigen positivity (odds ratio [OR], 5.28; 95% confidence interval [CI], 2.9 to 10.5) and intravenous drug injection (OR, 19; 95% CI, 3.4 to 149) were identified as factors associated with HCV Ab positivity. Spontaneous elimination of HCV infection was observed in 17.9% (7/39) of patients. DAA treatment was performed in 43 cases, and the overall sustained virologic response 12 (SVR12) rate for DAA treatment was 93% (40/43). CONCLUSION: A high HCV infection rate among HIV-infected MSM was observed in Japan. The DAA treatment response rate in patients with HIV/HCV co-infection was the high response rate.

Inhibition of CBP/ß-catenin signaling ameliorated fibrosis in cholestatic liver disease.

Chronic cholestatic liver diseases are characterized by injury of the bile ducts and hepatocytes caused by accumulated bile acids (BAs) and inflammation. Wnt/ß-catenin signaling is implicated in organ fibrosis; however, its role in cholestatic liver fibrosis remains unclear. Therefore, we explored the effect of a selective cAMP response element-binding protein-binding protein (CBP)/ß-catenin inhibitor, PRI-724, on murine cholestatic liver fibrosis. PRI-724 suppressed liver fibrosis induced by multidrug resistance protein 2 knockout (KO), bile duct ligation, or a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet; it also suppressed BA synthesis and macrophage infiltration. The expression of early growth response-1 (Egr-1), which plays a key role in BA synthesis, was increased in the hepatocytes of patients with cholestatic liver disease. PRI-724 inhibited Egr-1 expression induced by cholestasis, and adenoviral shEgr-1-mediated Egr-1 knockdown suppressed BA synthesis and fibrosis in DDC diet-fed mice, suggesting that PRI-724 exerts its effects, at least in part, by suppressing Egr-1 expression in hepatocytes. Hepatocyte-specific CBP KO in mice suppressed BA synthesis, liver injury, and fibrosis, whereas hepatocyte-specific KO of P300, a CBP homolog, exacerbated DDC-induced fibrosis. Intrahepatic Egr-1 expression was also decreased in hepatocyte-specific CBP-KO mice and increased in P300-KO mice, indicating that Egr-1 is located downstream of CBP/ß-catenin signaling. Conclusion: PRI-724 inhibits cholestatic liver injury and fibrosis by inhibiting BA synthesis in hepatocytes. These results highlight the therapeutic effect of CBP/ß-catenin inhibition in cholestatic liver diseases.

Safety, tolerability, and anti-fibrotic efficacy of the CBP/ß-catenin inhibitor PRI-724 in patients with hepatitis C and B virus-induced liver cirrhosis: An investigator-initiated, open-label, non-randomised, multicentre, phase 1/2a study.

BACKGROUND: We conducted an exploratory study to assess the safety tolerability, and anti-fibrotic effects of PRI-724, a CBP/ß-catenin inhibitor, in patients with hepatitis C virus (HCV)- and hepatitis B virus (HBV)-induced cirrhosis. METHODS: This multicentre, open-label, non-randomised, non-placebo-controlled phase 1/2a trial was conducted at three hospitals in Japan. Between July 27, 2018, and July 13, 2021, we enrolled patients with HCV- and HBV-induced cirrhosis classified as Child-Pugh (CP) class A or B. In phase 1, 15 patients received intravenous infusions of PRI-724 at escalating doses of 140, 280, and 380 mg/m2/4 h twice weekly for 12 weeks. In phase 2a, 12 patients received the recommended PRI-724 dose. The primary endpoints of phases 1 and 2a were the frequency and severity of adverse events and efficacy in treating cirrhosis based on liver biopsy. This study was registered at ClinicalTrials.gov (no. NCT03620474). FINDINGS: Three patients from phase 1 who received the recommended PRI-724 dose were evaluated to obtain efficacy and safety data in phase 2a. Serious adverse events occurred in three patients, one of which was possibly related to PRI-724. The most common adverse events were diarrhoea and nausea. PRI-724 did not decrease hepatic fibrosis with any statistical significance, either by ordinal scoring or measurement of collagen proportionate area at 12 weeks; however, we observed statistically significant improvements in liver stiffness, Model for End-stage Liver Disease score, and serum albumin level. INTERPRETATION: Intravenous administration of 280 mg/m2/4 h PRI-724 over 12 weeks was preliminarily assessed to be well tolerated; however, further evaluation of anti-fibrotic effects in patients with cirrhosis is warranted. FUNDING: AMED, Ohara Pharmaceutical.

Structural and spectroscopic characterization of CO inhibition of [NiFe]-hydrogenase from Citrobacter sp. S-77.

Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) is known to be a competitive inhibitor of O2-sensitive [NiFe]-hydrogenases. Although the activities of some O2-tolerant [NiFe]-hydrogenases are unaffected by CO, the partially O2-tolerant [NiFe]-hydrogenase from Citrobacter sp. S-77 (S77-HYB) is inhibited by CO. In this work, the CO-bound state of S77-HYB was characterized by activity assays, spectroscopic techniques and X-ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier-transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Šresolution revealed that CO binds weakly to the nickel ion in the Ni-Fe active site of S77-HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]-hydrogenases.

Nicotinamide mononucleotide production by fructophilic lactic acid bacteria.

Nicotinamide mononucleotide (NMN), an intermediate in nicotinamide adenine dinucleotide biosynthesis, is recently attracting much attention for its pharmacological and anti-aging efficacies. However, current commercial products containing NMN are very high-priced because efficient and facile methods for industrial NMN production are limited. In this study, aiming for its nutraceutical application, we attempted to screen lactic acid bacteria for intracellular and/or extracellular NMN production. Using a bioassay system with an auxotrophic yeast that requires nicotinamide riboside (NR; dephosphorylated NMN), three candidates were obtained from a library of 174 strains of facultative anaerobic lactic acid bacteria. All three candidates belonged to the genus Fructobacillus and produced NR in the culture media (0.8-1.5 mg/l). Lactic acid bacteria of the genus Fructobacillus are known to use D-fructose as an electron acceptor in anaerobic lactic acid fermentation; addition of D-fructose to the medium caused intracellular accumulation of NMN and NR, but no extracellular production of these compounds was observed. Draft genome sequencing for one of the candidates suggested that nicotinamide phosphoribosyltransferase, which exists commonly in mammals but is less reported in microorganisms, is a key enzyme for NMN and NR production in the fructophilic bacteria.

Towards cryogenic neutron crystallography on the reduced form of [NiFe]-hydrogenase.

A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni-Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni-Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni-Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.

A prospective trial of vaccine to prevent hepatitis B virus reactivation after hematopoietic stem cell transplantation.

Hepatitis B virus (HBV) reactivation reportedly occurs frequently after hematopoietic stem cell transplantation (HSCT) in resolved HBV-infected patients. Here, 50 patients with resolved HBV infections and scheduled to undergo HSCT were enrolled; all subjects were vaccinated with three doses of hepatitis B vaccine 12 months after HSCT and the incidence of HBV reactivation was monitored. The patients' characteristics were: median age, 61 (34-72) years; male/female, 27/19; allogeneic/autologous, 40/6; bone marrow/peripheral blood stem cells/cord blood, 26/16/4. Of the 46 patients who underwent HSCT, 19 were excluded and did not make it to vaccination due to relapse of underlying disease, HBV reactivation within 12 months of HSCT, or transfer of patients. The remaining 27 were vaccinated 12 months after HSCT and monitored for 2 years. Six showed HBV reactivation, with a 2-year cumulative reactivation incidence of 22.2%; the same incidence was 27.3% only in allogeneic HSCT patients. Factors associated with HBV reactivation included the discontinuation of immunosuppressants (P = 0.0379) and baseline titers of antibody against hepatitis B surface antigen (P = 0.004). HBV reactivation with vaccination following HSCT could occur despite maintenance of serum anti-HBs at more than protective levels.

MazF activation causes ACA sequence-independent and selective alterations in RNA levels in Escherichia coli.

Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.

Cysteine SH and Glutamate COOH Contributions to [NiFe] Hydrogenase Proton Transfer Revealed by Highly Sensitive FTIR Spectroscopy.

A [NiFe] hydrogenase (H2 ase) is a proton-coupled electron transfer enzyme that catalyses reversible H2 oxidation; however, its fundamental proton transfer pathway remains unknown. Herein, we observed the protonation of Cys546-SH and Glu34-COOH near the Ni-Fe site with high-sensitivity infrared difference spectra by utilizing Ni-C-to-Ni-L and Ni-C-to-Ni-SIa photoconversions. Protonated Cys546-SH in the Ni-L state was verified by the observed SH stretching frequency (2505 cm-1 ), whereas Cys546 was deprotonated in the Ni-C and Ni-SIa states. Glu34-COOH was double H-bonded in the Ni-L state, as determined by the COOH stretching frequency (1700 cm-1 ), and single H-bonded in the Ni-C and Ni-SIa states. Additionally, a stretching mode of an ordered water molecule was observed in the Ni-L and Ni-C states. These results elucidate the organized proton transfer pathway during the catalytic reaction of a [NiFe] H2 ase, which is regulated by the H-bond network of Cys546, Glu34, and an ordered water molecule.

Reliability and validity of the Japanese version of the ADL-focused Occupation-based Neurobehavioural Evaluation (A-ONE J): Applying Rasch analysis methods.

BACKGROUND/OBJECTIVE: The ADL-focused Occupation-based Neurobehavioral Evaluation (A-ONE) can be used to evaluate both performances of activities of daily living (ADL) tasks and neurobehavioural problems that interfere with ADL task performance among clients with neurological disorders. Research studies have demonstrated acceptable psychometric properties of the original version of the A-ONE as well as the Rasch analysed version. The aim of this study was to examine the reliability and validity of the Japanese version of the A-ONE (A-ONE J). METHODS: Rasch analysis was performed on data obtained from eight different hospitals in Japan on performances of 150 individuals diagnosed with a stroke based on the functional independence (FI) scale items. The rating scale structure was investigated and internal validity and reliability were examined. Unidimensionality of the items was examined by mean square infit values and principal component analysis of residuals. The targeting between person ability and item difficulty was explored, as well as the separation reliability. Finally, psychometric values and item difficulty hierarchies obtained in this study were compared to the original Rasch analysis of the A-ONE. RESULTS: The rating scale structure might be improved by collapsing two categories twice (from five categories to three categories). Unidimensionality of the items was obtained for 20 items. Targeting was acceptable, and separation reliability for item calibrations was high and acceptable for people.Conclusion/limitations: This study provides important information regarding the possibilities for revising the ordinal A-ONE J FI Scale, converting it into a unidimensional scale. Further study with increased and more diverse sample is needed.

Programmed cell death ligand 1 (PD-L1) blockade attenuates metastatic colon cancer growth in cAMP-response element-binding protein (CREB)-binding protein (CBP)/ß-catenin inhibitor-treated livers.

Immune checkpoint blockade with specific antibodies can accelerate anti-tumor immunity, resulting in clinical responses in patients with various types of cancer. However, these antibodies achieve only partial tumor regression. Thus, a wide variety of treatment combinations based on programmed death-ligand 1 (PD-L1) pathway inhibition are under development to enhance such therapeutic effects. In this study, the effects of combination treatment using PRI-724, a selective inhibitor of CBP/ß-catenin, and an anti-PD-L1 antibody were examined in a mouse model of colon cancer liver metastasis. Mice were inoculated with SL4 colon cancer cells to produce metastatic liver tumors. The combination treatment resulted in regression of tumor growth, whereas monotherapy with each treatment individually failed to exhibit any anti-tumor activity. In addition, co-administration of the inhibitor and antibody induced CD8+CD44lowCD62Llow cells and interferon (IFN)-γ production in CD8+ T-cells in the liver compared with that in control mice. Administration of an anti-CD8 antibody mitigated the anti-tumor effects of the combined treatment of PRI-724 and anti-PD-L1 antibody. In conclusion, targeting CBP/ß-catenin, combined with PD-1/PD-L1 immune checkpoint blockade, shows potential as a new therapeutic strategy for treating liver metastasis during colon cancer.

Development of Hepatocellular Carcinoma During Nivolumab Treatment for Recurrent Non-Small Cell Lung Cancer: A Case Report.

Nivolumab, a monoclonal antibody targeting programmed cell death 1 (PD-1), is the standard second-line therapy for advanced non-small cell lung cancer (NSCLC). In the current immunotherapy era, it is often difficult to evaluate the therapeutic effect, disease progression, and pseudo-enlargement of the tumor or the emergence of another etiology. In the present report, we describe a 79-year-old patient with hepatocellular carcinoma (HCC) newly detected during nivolumab treatment for recurrent NSCLC. When the patient was 73 years old, he had suffered from NSCLC and received concurrent chemoradiotherapy comprising cisplatin and docetaxel, achieving a complete response. Six years after the chemoradiotherapy, the patient had multiple lung and hepatic lesions. We thus started the treatment with nivolumab for recurrent NSCLC. All those lesions responded to nivolumab over nine cycles. By contrast, a lesion was newly detected in the medial segment of left hepatic lobe, liver segment 4 (S4), and was gradually getting larger, as judged by computed tomographic scan. Liver biopsy revealed the growing lesion to be a well-differentiated HCC. Consequently, the patient was treated with radiofrequency ablation to HCC, while nivolumab treatment was continued for NSCLC. Immunohistochemical analysis of the HCC specimens revealed nuclear accumulation of ß-catenin compared with normal liver cells and undetectable expression of program death ligand 1 (PD-L1). Such expression profiles of ß-catenin and PD-L1 in HCC may be responsible for the resistance against nivolumab treatment. Immunohistochemical features of the biopsy specimens may be predictive of the effectiveness of the immunotherapy in HCC.

New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates.

Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox-dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non-invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H2 and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase-mediated reactions simultaneously. Using the proposed system, we confirmed that H2 (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D2 /H2 O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (kout ) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.