Novel Use of Time-Resolved Magnetic Resonance Angiography to Evaluate Retrograde Lumbar Vein Flow and Epidural Venous Plexus Enhancement in Chronic Headache Patients With and Without Nutcracker Physiology.

OBJECTIVE: Compression of the left renal vein by the superior mesenteric artery, known as nutcracker phenomenon (NCP), can cause retrograde flow and congestion in communicating venous systems. It has recently been speculated that NCP can result in retrograde flow and congestion of the lumbar veins and epidural venous plexus (EVP), thereby affecting the central nervous system. This study describes the novel use of time-resolved magnetic resonance angiography (trMRA) to evaluate for retrograde left second lumbar vein (L2LV) flow and early EVP enhancement in patients with chronic daily headache (CDH) with and without NCP. METHODS: A retrospective analysis was performed of 31 patients with CDH (27 females and 4 males; median age, 38 years [range, 18-63 years]) who underwent trMRA centered over the L2LV to evaluate the direction of blood flow and presence of early EVP enhancement from May 2020 to March 2022. Descriptive statistics were performed, and anatomic associations were analyzed in patients with and without retrograde L2LV flow and early EVP enhancement. The accuracy of magnetic resonance imaging findings in detecting these flow patterns was also assessed. RESULTS: Patients with NCP who demonstrated narrowing of the left renal vein, a positive beak sign ( P = 0.052), decreased aortomesenteric distance ( P = 0.038), and decreased SMA angle demonstrated increased rates of retrograde L2LV flow and early EVP enhancement. A positive beak sign was 83% specific, and an aortomesenteric distance of ≤6.5 mm was 61% sensitive and 83% specific for identifying retrograde L2LV flow with early regional EVP enhancement in patients with CDH. CONCLUSIONS: Retrograde L2LV flow with early EVP enhancement in CDH patients can be effectively evaluated using trMRA and was seen with greater propensity in those patients with NCP.

Hepatocellular carcinoma radiation segmentectomy treatment intensification prior to liver transplantation increases rates of complete pathologic necrosis: an explant analysis of 75 tumors.

PURPOSE: To verify the correlation between yttrium-90 glass microsphere radiation segmentectomy treatment intensification of hepatocellular carcinoma (HCC) and complete pathologic necrosis (CPN) at liver transplantation. METHODS: A retrospective, single center, analysis of patients with HCC who received radiation segmentectomy prior to liver transplantation from 2016 to 2021 was performed. The tumor treatment intensification cohort (n = 38) was prescribed radiation segmentectomy as per response recommendations identified in a previously published baseline cohort study (n = 37). Treatment intensification and baseline cohort treatment parameters were compared for rates of CPN. Both cohorts were then combined for an overall analysis of treatment parameter correlation with CPN. RESULTS: Sixty-three patients with a combined 75 tumors were analyzed. Specific activity, dose, and treatment activity were significantly higher in the treatment intensification cohort (all p < 0.01), while particles per cubic centimeter of treated liver were not. CPN was achieved in 76% (n = 29) of tumors in the treatment intensification cohort compared to 49% (n = 18) in the baseline cohort (p = 0.013). The combined cohort CPN rate was 63% (n = 47). ROC analysis showed that specific activity ≥ 327 Bq (AUC 0.75, p < 0.001), dose ≥ 446 Gy (AUC 0.69, p = 0.005), and treatment activity ≥ 2.55 Gbq (AUC 0.71, p = 0.002) were predictive of CPN. Multivariate logistic regression demonstrated that a specific activity ≥ 327 Bq was the sole independent predictor of CPN (p = 0.013). CONCLUSION: Radiation segmentectomy treatment intensification for patients with HCC prior to liver transplantation increases rates of CPN. While dose strongly correlated with pathologic response, specific activity was the most significant independent radiation segmentectomy treatment parameter associated with CPN.

Radiation Segmentectomy for the Treatment of Solitary Hepatocellular Carcinoma: Outcomes Compared with Those of Surgical Resection.

PURPOSE: To investigate the outcomes of radiation segmentectomy (RS) versus standard-of-care surgical resection (SR). MATERIALS AND METHODS: A multisite, retrospective analysis of treatment-naïve patients who underwent either RS or SR was performed. The inclusion criteria were solitary hepatocellular carcinoma ≤8 cm in size, Eastern Cooperative Oncology Cohort performance status of 0-1, and absence of macrovascular invasion or extrahepatic disease. Target tumor and overall progression, time to progression (TTP), and overall survival rates were assessed. Outcomes were censored for liver transplantation. RESULTS: A total of 123 patients were included (RS, 57; SR, 66). Tumor size, Child-Pugh class, albumin-bilirubin score, platelet count, and fibrosis stage were significantly different between cohorts (P ≤ .01). Major adverse events (AEs), defined as grade ≥3 per the Clavien-Dindo classification, occurred in 0 patients in the RS cohort vs 13 (20%) patients in the SR cohort (P < .001). Target tumor progression occurred in 3 (5%) patients who underwent RS and 5 (8%) patients who underwent SR. Overall progression occurred in 19 (33%) patients who underwent RS and 21 (32%) patients who underwent SR. The median overall TTP was 21.9 and 29.4 months after RS and SR, respectively (95% confidence interval [CI], 15.5-28.2 and 18.5-40.3, respectively; P = .03). Overall TTP subgroup analyses showed no difference between treatment cohorts with fibrosis stages 3-4 (P = .26) and a platelet count of <150 × 109/L (P = .29). The overall progression hazard ratio for RS versus SR was not significant per the multivariate Cox regression analysis (1.16; 95% CI, 0.51-2.63; P = .71). The median overall survival was not reached for either of the cohorts. Propensity scores were calculated but were too dissimilar for analysis. CONCLUSIONS: RS and SR were performed in different patient populations, which limits comparison. RS approached SR outcomes, with a lower incidence of major AEs, in patients who were not eligible for hepatectomy.

Natural History of Incidental Enhancing Nodules on Cone-Beam Computed Tomography during Transarterial Therapy of Hepatocellular Carcinoma.

PURPOSE: To evaluate the natural history of incidental enhancing nodules (IENs) on contrast-enhanced cone-beam computed tomography (CT) during transarterial treatment of hepatocellular carcinoma (HCC). MATERIAL AND METHODS: A single-center retrospective analysis of 100 patients with HCC who underwent contrast-enhanced cone-beam CT prior to transarterial treatment from August 2015 to June 2019 was performed. Inclusion criteria were patients with segmental distribution sublobar HCC, contrast-enhanced cone-beam CT of the target lesion and nontarget liver parenchyma, and follow-up cross-sectional imaging. Patients with IENs ≥3 mm that did not meet imaging criteria for HCC were analyzed. Exclusion criteria included biphenotypic tumors and IEN present inside the treated area of the liver. RESULTS: Fifty-six patients demonstrated 154 IENs on contrast-enhanced cone-beam CT, of which 13 IENs (8.5%) progressed to HCC. The mean primary tumor size was 29 mm (range: 10.2-189 mm). Ten patients had ≥4 IENs, and 46 patients had 1-3 IENs. The mean IEN size was 6.8 mm (range: 3.0-16.3 mm). The median follow-up interval after contrast-enhanced cone-beam CT was 282 days (interquartile range: 143-522). Increased alpha-fetoprotein before treatment (≥15.5 ng/mL, P = .035), having ≥4 IENs (P = .020), and hepatitis C virus (P = .015) were significantly correlated with IEN progression to HCC. No statistically significant differences were identified in baseline neutrophil-to-lymphocyte ratio, targeted HCC characteristics (size, macrovascular invasion, infiltrative pattern, enhancement pattern, and satellite lesions), and IEN size between those with IEN progression to HCC and those without. CONCLUSIONS: Most IENs of ≥3 mm on contrast-enhanced cone-beam CT in patients with segmental distribution sublobar HCC do not progress to HCC. Patients with segmental distribution sublobar HCC with ≥4 IENs, alpha-fetoprotein elevation (≥15.5 ng/mL), or hepatitis C virus have an increased risk of IEN progression to HCC.

Pathologic Response of Hepatocellular Carcinoma Treated with Yttrium-90 Glass Microsphere Radiation Segmentectomy Prior to Liver Transplantation: A Validation Study.

PURPOSE: To evaluate the pathologic outcomes of hepatocellular carcinoma (HCC) treated with Yttrium-90 radiation segmentectomy using glass microspheres prior to liver transplantation and explore parameters associated with pathologic necrosis. MATERIALS AND METHODS: A single-institution retrospective analysis of HCC patients who received radiation segmentectomy prior to liver transplantation from November 2016 to May 2020 was performed. Patients were included if the treatment angiosome encompassed the entire tumor and could be correlated with available gross pathology. Archived histology slides were reviewed for percentage of pathologic necrosis. Thirty-three patients with 37 tumors were evaluated. The median tumor size was 2.3 cm (range, 1-6.7 cm). RESULTS: All tumors received a single treatment. The median time from radiation segmentectomy to transplantation was 206 days (range, 58-550 days). Objective response per Modified Response Evaluation Criteria in Solid Tumors (mRECIST) was 92% (complete response, 76%; partial response, 16%). A total of 68% (n = 25) of tumors demonstrated ≥99% pathologic necrosis. Complete pathologic necrosis was present in 53% and 75% of tumors treated with >190 Gy (n = 18) and >500 Gy (n = 8) single-compartment Medical Internal Radiation Dose, respectively. Complete response per mRECIST, posttreatment angiosome T1 hypointensity, dose >190 Gy, microsphere specific activity >297 Bq, and a longer time between treatment and transplant were associated with ≥99% tumor necrosis (P < .05). No posttransplant tumor recurrences occurred within a median follow-up of 604 days (range, 138-1,223 days). CONCLUSIONS: Radiation segmentectomy can serve as an ablative modality for the treatment of HCC prior to liver transplant.

Increasing Yttrium-90 Dose Conformality Using Proximal Radioembolization Enabled by Distal Angiosomal Truncation for the Treatment of Hepatic Malignancy.

PURPOSE: To evaluate safety and feasibility of improving radiation dose conformality via proximal radioembolization enabled by distal angiosomal truncation where selective administration was not practical. MATERIALS AND METHODS: Hepatic malignancies treated via angiosomal truncation between January 2017 and March 2019 were retrospectively evaluated. Thirty-three patients (8 women, 25 men; mean age, 62.2 y; range, 36-78 y) underwent 39 treatments. Of treatments, 74.3% (n = 29) were for hepatocellular carcinomas, 10.2% (n = 4) were for cholangiocarcinomas, and 15.4% (n = 6) were for metastatic tumors (1 colorectal adenocarcinoma, 1 pancreatic adenocarcinoma, 3 melanomas, and 1 endometroid carcinoma). Truncation was achieved using temporary embolic devices including a microvascular plug, detachable coil, gelatin slurry, and balloon microcatheter, after which proximal radioembolization was performed. Range of treatment activity was 0.47-5.75 GBq. Technetium-99m macroaggregated albumin and bremsstrahlung single photon emission computed tomography (CT)/CT threshold analysis was conducted to delineate and compare distribution of activity within the treatment angiosome before and after radioembolization. RESULTS: Dosimetric analysis of 14 patients demonstrated a significant reduction in nontarget liver radiation exposure at 5, 20, and 40% thresholds (P = .002, P = .001, and P = .008, respectively). There were no grade 3 or higher adverse events. There was no significant change in Albumin-Bilirubin grade and Eastern Cooperative Oncology Group Performance Status (P = .09 and P = .74) before and 3 months after the procedure. Truncated arteries were patent on subsequent angiography in 11 cases and on MR angiography or CT angiography in 38 of 39 cases. CONCLUSIONS: Proximal radioembolization enabled by distal angiosomal truncation is safe and decreases nontarget parenchymal radioembolization dose in cases not amenable to selective administration.

Reisolation and NMR characterization of the satratoxins G and H.

The exquisitely cytotoxic macrolides, satratoxins G and H, have been reisolated from a solvent extract of a rice culture inoculated with Stachybotrys chartarum to be used as high-purity reference compounds for analytical analyses. Extensive chromatographic separation realized the compounds that were fully recharacterized in two solvents by 1D- and 2D-NMR spectroscopy, revealing some discrepancies in the nuclear magnetic resonance (NMR) data as compared with the previously reported values found in the literature. Detailed spectra are provided in order to aid future identification and dereplication.

Both Chemical and Non-Chemical Steps Limit the Catalytic Efficiency of Family 4 Glycoside Hydrolases.

The glycoside hydrolase family 4 (GH4) α-galactosidase from Citrobacter freundii (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD+ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived ßlg value on V/ K for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr2+, Y3+, and Mn2+, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured D V/ K KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-galactopyranoside are closer to unity than the measured effects on 4-fluorophenyl α-d-galactopyranoside, irrespective of the site of isotopic substitution and of the metal cation activator. These observations are consistent with hydride transfer at C-3 to the on-board NAD+, deprotonation at C-2, and a non-chemical step contributing to the virtual TS for V/ K.

Transition Metal Ions Promote the Bioavailability of Hydrophobic Therapeutics: Cu and Zn Interactions with RNA Polymerase†I Inhibitor CX5461.

Low aqueous solubility is a major barrier to the clinical application of otherwise promising drug candidates. We demonstrate that this issue can be resolved in medicinal molecules containing potential ligating groups, through the addition of labile transition-metal ions. Incubation of the chemotherapeutic CX5461 with Cu2+ or Zn2+ enables solubilization at neutral pH but does not affect intrinsic cytotoxicity. Spectroscopic and computational studies demonstrate that this arises from coordination to the pyrazine functionality of CX5461 and may involve bidentate coordination at physiological pH.

CT-guided Native Medical Renal Biopsy: Cortical Tangential versus Non-Tangential Approaches-A Comparison of Efficacy and Safety.

Purpose To review a single-center experience with the cortical tangential approach during computed tomography (CT)-guided native medical renal biopsy and to evaluate its efficacy and safety compared with those of a non-cortical tangential approach. Materials and Methods This retrospective study received institutional review board approval, with a waiver of the HIPAA requirement for informed consent. The number of cores, glomeruli, and complications were reviewed in 431 CT-guided medical renal biopsies performed between July 2007 and September 2015. A biopsy followed a cortical tangential approach if the needle path was parallel to the renal cortical surface, at a depth closer to the renal capsule than the renal pelvic fat. A sample was considered adequate if the biopsy yielded at least 10 glomeruli at light microscopy, one glomerulus at immunofluorescence microscopy, and one glomerulus at electron microscopy. The χ2 test, the t test, the Mann-Whitney test, and logistic regression modeling of sample adequacy were performed. Results One hundred fifty-six (36%) of 431 biopsies were performed with the cortical tangential approach. More cores were obtained for the cortical tangential group (2.6 vs 2.4, P = .001); biopsy needle gauge was not significantly different (P = .076). More adequate samples were obtained in the cortical tangential group (66.7% vs 49.8%, P = .001), with more glomeruli (23 vs 16, P = .014). Results were significant after controlling for needle gauge and number of cores (P = .008). The cortical tangential group had fewer complications (1.9% vs 7.3%, P = .018). Conclusion The cortical tangential approach, when applied to CT-guided native medical renal biopsies, results in higher rates of sample adequacy and lower rates of postprocedural complications. © RSNA, 2016.

CF3 Derivatives of the Anticancer Ru(III) Complexes KP1019, NKP-1339, and Their Imidazole and Pyridine Analogues Show Enhanced Lipophilicity, Albumin Interactions, and Cytotoxicity.

The Ru(III) complexes indazolium [trans-RuCl4(1H-indazole)2] (KP1019) and sodium [trans-RuCl4(1H-indazole)2] (NKP-1339) are leading candidates for the next generation of metal-based chemotherapeutics. Trifluoromethyl derivatives of these compounds and their imidazole and pyridine analogues were synthesized to probe the effect of ligand lipophilicity on the pharmacological properties of these types of complexes. Addition of CF3 groups also provided a spectroscopic handle for (19)F NMR studies of ligand exchange processes and protein interactions. The lipophilicities of the CF3-functionalized compounds and their unsubstituted parent complexes were quantified by the shake-flask method to give the distribution coefficient D at pH 7.4 (log D7.4). The solution behavior of the CF3-functionalized complexes was characterized in phosphate-buffered saline (PBS) using (19)F NMR, electron paramagnetic resonance (EPR), and UV-vis spectroscopies. These techniques, along with fluorescence competition experiments, were also used to characterize interactions with human serum albumin (HSA). From these studies it was determined that increased lipophilicity correlates with reduced solubility in PBS but enhancement of noncoordinate interactions with hydrophobic domains of HSA. These protein interactions improve the solubility of the complexes and inhibit the formation of oligomeric species. EPR measurements also demonstrated the formation of HSA-coordinated species with longer incubation. (19)F NMR spectra show that the trifluoromethyl complexes release axial ligands in PBS and in the presence of HSA. In vitro testing showed that the most lipophilic complexes had the greatest cytotoxic activity. Addition of CF3 groups enhances the activity of the indazole complex against A549 nonsmall cell lung carcinoma cells. Furthermore, in the case of the pyridine complexes, the parent compound was inactive against the HT-29 human colon carcinoma cell line but showed strong cytotoxicity with CF3 functionalization. Overall, these studies demonstrate that lipophilicity may be a determining factor in the anticancer activity and pharmacological behavior of these types of Ru(III) complexes.

Abscopal downstaging of intermediate stage hepatocellular via combination cryoablation and immunotherapy with complete pathologic response.

The abscopal effect is a rare phenomenon characterized by disease regression in distant sites after tumoral locoregional therapy. Locoregional therapy, such as cryoablation, can induce an antitumor immunological response, potentially improving outcomes in cancer patients receiving immunotherapy. This report describes a patient with multifocal hepatocellular carcinoma who progressed through multiple locoregional therapies, was initially unresponsive to immunotherapy, and later achieved rapid and sustained disease regression with a combination cryoablation and immunotherapy. A 5-year sustained complete tumor response successfully bridged to liver transplantation.

Histologic Findings of Sinusoidal Dilatation and Congestion in Liver Grafts Do Not Correlate with Hepatic Venous Anastomotic Gradients.

PURPOSE: Hepatic venous transplant anastomotic pressure gradient measurement and transjugular liver biopsy are commonly used in clinical decision-making in patients with suspected anastomotic hepatic venous outflow obstruction. This investigation aimed to determine if sinusoidal dilatation and congestion on histology are predictive of hepatic venous anastomotic outflow obstruction, and if it can help select patients for hepatic vein anastomosis stenting. MATERIALS AND METHODS: This is a single-center retrospective study of 166 transjugular liver biopsies in 139 patients obtained concurrently with transplant venous anastomotic pressure gradient measurement. Demographic characteristics, laboratory parameters, procedure and clinical data, and histology of time-zero allograft biopsies were analyzed. RESULTS: No relationship was found between transplant venous anastomotic pressure gradient and sinusoidal dilatation and congestion (P = 0.92). Logistic regression analysis for sinusoidal dilatation and congestion confirmed a significant relationship with reperfusion/preservation injury and/or necrosis of the allograft at time-zero biopsy (OR 6.6 [1.3-33.1], P = 0.02). CONCLUSION: There is no relationship between histologic sinusoidal dilatation and congestion and liver transplant hepatic vein anastomotic gradient. In this study group, sinusoidal dilatation and congestion is a nonspecific histopathologic finding that is not a reliable criterion to select patients for venous anastomosis stenting.

Ablative radioembolization of hepatocellular carcinoma with total arterial supply originating from the superior adrenal artery achieved complete pathologic necrosis.

Exclusively extrahepatic arterial perfusion to previously untreated, early-stage, hepatocellular carcinoma (HCC) is uncommon. We present a case of right superior adrenal artery completely supplying a subcapsular HCC in a 68-year-old male. The extrahepatic arterial territory targeted excluded structures potentially vulnerable to radiation, which allowed safe ablative Y-90 radioembolization that achieved complete pathological necrosis.

Spatial characterization of redox processes and speciation of Ru(III) anticancer complexes by 19F magnetic resonance imaging.

The application of CF3-labeled Ru(III) anticancer complexes to magnetic resonance (MR) imaging of tumour tissues is demonstrated. By combining anatomical chemical-shift selective (CHESS) imaging with 19F chemical-shift imaging (CSI) MR methods, we show that oxidation states and ligand-exchange processes of the complexes can be spatially encoded. Measurements on different tissue models, including a human breast adenocarcinoma tumour, validate the application of these complexes as MR theranostics for the sensing and targeting of hypoxia.

A secondary daily persistent headache from onset with underlying nutcracker physiology and spinal epidural venous congestion: case series with lumbar vein embolization as a therapeutic approach.

The authors have published on a unique subset of patients whose headaches worsened in the Trendelenburg position and who on time-resolved MR angiography demonstrated left renal vein compression (nutcracker physiology) with retrograde left second lumbar vein (L2LV) flow and regional spinal epidural venous plexus (EVP) congestion. We hypothesized that the spinal EVP congestion subsequently causes a secondary congestion of the cerebral venous system, which then leads to an elevation of CSF pressure above that individuals CSF pressure set point. This results in a daily headache from onset. Thus, eliminating the spinal EVP could conceivably improve or eliminate the manifested headache syndrome. We now present a case series of four patients with long-term follow-up utilizing lumbar vein coil embolization as a new therapeutic approach. In each patient, the MR angiography findings were verified by catheter-based venography. Treatment consisted of endovascular embolization of the second lumbar vein. Four patients have had coil embolization of which three are 1 year or longer from their procedure while one is 10 months posttreatment. All patients were women. Duration of daily headache prior to embolization ranged from 4 to 8 years. Post-embolization: Three patients are either headache free or 90-95% improved with substantial pain free time. There were no procedure-related complications. Our results suggest that embolization of L2LV in a specific patient population with nutcracker physiology may substantially improve head pain issues. This is a minimally invasive outpatient technique with no apparent side effects.

Outcomes of Radiation Segmentectomy for Hepatocellular Carcinoma in Patients with Non-Alcoholic Fatty Liver Disease versus Chronic Viral Hepatitis.

Purpose: To compare the outcomes of radiation segmentectomy for early-stage hepatocellular carcinoma (HCC) in patients with non-alcoholic fatty liver disease (NAFLD) versus hepatitis C virus (HCV). Materials and Methods: A retrospective analysis of consecutive patients with NAFLD- or HCV-related HCC treated with radiation segmentectomy from 01/2017-06/2022 was performed. Eligibility criteria included solitary tumor ≤8 cm or up to 3 HCC ≤3 cm, ECOG 0-1, and absence of vascular invasion or extrahepatic spread. Imaging best response was assessed per modified Response Evaluation Criteria in Solid Tumors. Target tumor and overall progression, time-to-progression (TTP), and overall survival (OS) were calculated. All outcomes were censored for liver transplantation (LT). Complete pathologic response (CPN) was assessed in patients who underwent LT. Results: Of 142 patients included (NAFLD: 61; HCV: 81), most had cirrhosis (NAFLD: 87%; HCV: 86%) and small tumors (median size NAFLD: 2.3 cm; HCV: 2.5 cm). Patients with NAFLD had higher BMI (p<0.001) and worse ALBI scores (p=0.003). Patients with HCV were younger (p<0.001) and had higher AFP levels (p=0.034). Median radiation dose (NAFLD: 508 Gy; HCV: 452 Gy) and specific activity (NAFLD: 700 Bq; HCV: 698 Bq) were similar between cohorts. Objective response was 100% and 97% in the NAFLD and HCV cohorts, respectively. Target tumor progression occurred in 1 (2%) NAFLD and 8 (10%) HCV patients. Target tumor TTP was not met for either cohort. Overall progression occurred in 23 (38%) NAFLD and 39 (48%) HCV patients. Overall TTP was 17.4 months (95% CI 13.5-22.2) in NAFLD and 13.5 months (95% CI 0.4-26.6) in HCV patients (p=0.86). LT was performed in 27 (44%) NAFLD and 33 (41%) HCV patients, with a CPN rate of 63% and 54%, respectively. OS was not met in the NAFLD cohort and was 53.9 months (95% CI 32.1-75.7) in the HCV cohort (p=0.15). Conclusion: Although NAFLD and HCV are associated with different mechanisms of liver injury, patients with early-stage HCC treated with radiation segmentectomy achieve comparable outcomes.

Transition state analysis of Vibrio cholerae sialidase-catalyzed hydrolyses of natural substrate analogues.

A series of isotopically labeled natural substrate analogues (phenyl 5-N-acetyl-α-d-neuraminyl-(2→3)-ß-d-galactopyranosyl-(1→4)-1-thio-ß-d-glucopyranoside; Neu5Acα2,3LacßSPh, and the corresponding 2→6 isomer) were prepared chemoenzymatically in order to characterize, by use of multiple kinetic isotope effect (KIE) measurements, the glycosylation transition states for Vibrio cholerae sialidase-catalyzed hydrolysis reactions. The derived KIEs for Neu5Acα2,3LacßSPh for the ring oxygen ((18)V/K), leaving group oxygen ((18)V/K), C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.029 ± 0.002, 0.983 ± 0.001, 1.034 ± 0.002, and 1.043 ± 0.002, respectively. In addition, the KIEs for Neu5Acα2,6ßSPh for C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.021 ± 0.001 and 1.049 ± 0.001, respectively. The glycosylation transition state structures for both Neu5Acα2,3LacßSPh and Neu5Acα2,6LacßSPh were modeled computationally using the experimental KIE values as goodness of fit criteria. Both transition states are late with largely cleaved glycosidic bonds coupled to pyranosyl ring flattening ((4)H(5) half-chair conformation) with little or no nucleophilic involvement of the enzymatic tyrosine residue. Notably, the transition state for the catalyzed hydrolysis of Neu5Acα2,6ßSPh appears to incorporate a lesser degree of general-acid catalysis, relative to the 2,3-isomer.

A direct NMR method for the measurement of competitive kinetic isotope effects.

We present a technique that uses (13)C NMR spectroscopy to measure kinetic isotope effects on the second-order rate constant (k(cat)/K(m)) for enzyme-catalyzed reactions. Using only milligram quantities of isotopically labeled substrates, precise competitive KIEs can be determined while following the ongoing reaction directly in a NMR spectrometer. Our results for the Vibrio cholerae sialidase-catalyzed hydrolysis of natural substrate analogs support a concerted enzymatic transition state for these reactions.