Anatomical and clinical factors associated with infrapopliteal arterial bypass outcomes in patients with chronic limb-threatening ischemia.

The aim of this study was to identify anatomical and clinical factors associated with limb-based patency (LBP) loss, major adverse limb events (MALEs), and poor amputation-free survival (AFS) after an infrapopliteal arterial bypass (IAB) surgery according to the Global Limb Anatomic Staging System. A retrospective analysis of patients undergoing IAB surgery between January 2010 and December 2021 at a single institution was performed. Two-year AFS, freedom from LBP loss, and freedom from MALEs were assessed using the Kaplan-Meier method. Anatomical and clinical predictors were assessed using multivariate analysis. The total number of risk factors was used to calculate risk scores for subsequent categorization into low-, moderate-, and high-risk groups. IABs were performed on 103 patients. The rates of two-year freedom from LBP loss, freedom from MALEs, and AFS were 71.3%, 76.1%, and 77.0%, respectively. The multivariate analysis showed that poor run-off beyond the ankle and a bypass vein caliber of < 3 mm were significantly associated with LBP loss and MALEs. Moreover, end-stage renal disease, non-ambulatory status, and a body mass index of < 18.5 were significantly associated with poor AFS. The rates of freedom from LBP loss and MALEs and the AFS rate were significantly lower in the high-risk group than in the other two groups (12-month low-risk rates: 92.2%, 94.8%, and 94.4%, respectively; 12-month moderate-risk rates: 58.6%, 84.6%, and 78.3%, respectively; 12-month high-risk rates: 11.1%, 17.6%, and 56.2%, respectively; p < 0.001, p < 0.001, and p < 0.001, respectively). IAB is associated with poor clinical outcomes in terms of LBP, MALEs, and AFS in high-risk patients. Risk stratification based on these predictors is useful for long-term prognosis.

Efficacy of Fusion Imaging in Endovascular Revascularization of the Superficial Femoral Artery.

BACKGROUND: The demand for endovascular revascularization (ER) to treat peripheral artery disease (PAD) has steadily increased. However, ER comes at the cost of increased contrast and radiation exposure, particularly in more complex cases. Fusion imaging is a new technology that may address these issues. The purpose of this study was to evaluate the efficacy of fusion imaging in ER of the superficial femoral artery (SFA). METHODS: Patients with PAD undergoing ER of the SFA from February 2016 to July 2020 were retrospectively evaluated. A group of patients treated using fusion imaging was compared with a control group treated without fusion imaging. The primary end points were the contrast dose, fluoroscopy time, radiation dose, and operative time. RESULTS: A total of 51 patients (fusion group, n = 26; control group, n = 25) underwent ER during the study period. Significantly lower iodinated contrast doses were observed in the fusion than in the control group (56.1 ± 23.7 vs. 87.9 ± 44.9 mL; P = 0.003), as well as significantly shorter fluoroscopy times (21.2 ± 11.1 vs. 44.9 ± 31.4 min; P = 0.001), lower radiation exposure (29.9 ± 8.9 vs. 122.2 ± 223.1 mGy; P = 0.04), and shorter operative times (88.3 ± 32.1 vs. 126.1 ± 66.8 min; P = 0.013). CONCLUSIONS: The use of fusion imaging technology during ER of the SFA can significantly reduce the contrast dose, fluoroscopy time, radiation dose, and operative time.

Early evaluation of the infrainguinal revascularization strategy selection tool of the Global Vascular Guidelines for chronic limb-threatening ischemia patients.

OBJECTIVE: The Global Vascular Guidelines (GVG) propose a novel Global Anatomic Staging System (GLASS) with the Wound, Ischemia, and foot Infection (WIfI) classification system as a clinical decision-making tool for interventions in chronic limb-threatening ischemia (CLTI). We assessed the validity of clinical staging and the relationship between the treatments recommended by the GVG and the outcomes of the actual procedures. METHODS: This retrospective, single-center, observational study included 117 patients with CLTI undergoing infrainguinal revascularization in our hospital between 2015 and 2019. Of those patients, 55 underwent open bypass (OB) and 62 underwent endovascular revascularization (EVR). Femoropopliteal, infrapopliteal, and inframalleolar GLASS grades were assigned based on angiographic images. These grades were combined to determine the revascularization strategy recommended by the GVG: "endovascular," "indeterminate," and "open bypass." The indeterminate category includes three subcategories: GLASS stage III, WIfI stage 2; GLASS stage II, WIfI stage 3; and GLASS stage II, WIfI stage 4. For the purposes of this study, we labeled these subcategories A, B, and C, respectively. The primary outcome was the correlation between the revascularization strategies recommended by the GVG and the actual procedures performed. The relationships between the actual procedures and overall survival, limb salvage, and patency were also examined. RESULTS: The femoropopliteal and infrapopliteal GLASS grades were higher in the OB group. EVR was performed more often for GLASS stages I and II and was more often classified as indeterminate B and C, whereas OB was performed more often in GLASS stage III and was more often classified as indeterminate A. There were no statistically significant differences in the inframalleolar/pedal disease descriptor or in the 30-day postoperative complication rates between the two groups. In higher GLASS stages, the technical success rate of EVR was lower, and lesion complexity was more severe. Patients for whom the recommended strategy according to the GVG would have been OB but who underwent EVR were associated with low limb salvage and patency rates. CONCLUSIONS: The GVG provide good guidance for the selection of the revascularization strategy. When the GVG indicate OB, it should be the treatment of choice, rather than EVR, for patients who are fit to undergo the procedure.

Bicycle exercise training improves ambulation in patients with peripheral artery disease.

OBJECTIVE: Exercise training has multiple beneficial effects in patients with arteriosclerotic diseases; however, the exact underlying mechanisms of the effects are not completely understood. This study aimed to evaluate the effectiveness of a supervised exercise program in improving gait parameters, including the variability and walking performance of lower limb movements, in patients with peripheral artery disease (PAD) and intermittent claudication (IC). METHODS: Sixteen patients with a history of PAD and IC were recruited for this study, and they completed a 3-month supervised bicycle exercise program. The ankle-brachial index and responses to quality of life (QOL) questionnaires were evaluated. Near-infrared spectroscopy was also performed to determine the hemoglobin oxygen saturation in the calf. Patients' kinematics and dynamics, including joint range of motion and muscle tension, were evaluated using an optical motion capture system. Computed tomography images of each muscle were assessed by manual outlining. Data were collected before and after the supervised bicycle exercise program, and differences were analyzed. RESULTS: Significant differences were not found in step length, ankle-brachial index, and hemoglobin oxygen saturation before and after the supervised bicycle exercise program; however, IC distance (P = .034), maximum walking distance (P = .006), and all QOL questionnaire scores (P < .001) showed significant improvement. Hip range of motion (P = .035), maximum hip joint torque (right, P = .031; left, P = .044), maximum tension of the gluteus maximus muscle (right, P = .044; left, P = .042), and maximum hip joint work (right, P = .048; left, P = .043) also significantly decreased bilaterally. Computed tomography images showed a significant increase in the cross-sectional area of the abdominal, trunk, and thigh muscles but not in that of the lower leg muscles after the supervised exercise program intervention. CONCLUSIONS: In this study, bicycle exercise training improved the QOL and walking distance and decreased hip movement. The results showed that bicycling might be as useful as walking in patients with PAD.

A perioperative strategy for abdominal aortic aneurysm in patients with chronic renal insufficiency.

PURPOSE: The outcomes of open surgical repair (OR) or endovascular aneurysm repair (EVAR) for abdominal aortic aneurysm (AAA) are favorable; however, pre-existing chronic renal insufficiency (CRI) is considered to be a risk factor that can affect the long-term outcome. We evaluated our surgical strategy for AAA in patients with CRI by analyzing their pre- and postoperative renal function. METHODS: We conducted a retrospective chart review of CRI patients who underwent OR (n = 28) or EVAR (n = 31) for infra-renal AAA in our institution between 2009 and 2013. Our operative strategy included pre- and postoperative adequate hydration, postoperative diuretics and low-dose dopamine for both groups, intravascular ultrasonography and carbon dioxide angiography to reduce the amount of contrast media needed in the EVAR group, and occasional intraoperative mannitol for the OR group. RESULTS: The preoperative estimated glomerular filtration rate (eGFR) increased significantly in the postoperative period and remained similar 6 months later in both groups, without any difference in changes between the groups. In-hospital postoperative complications included leg occlusion in one EVAR patient. There were no complication-related deaths in either group. CONCLUSIONS: Postoperative renal function was similar after the two approaches, indicating that both procedures could be performed safely using our strategy for patients with CRI.

Eukaryotic translation elongation factor 1A induces anoikis by triggering cell detachment.

Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing ß1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage.

High-resolution inventory of Japanese anthropogenic mercury emissions.

Heavy metals like mercury that are emitted into the environment remain there indefinitely, posing a long-term threat to both the environment and human health. Elemental mercury is volatile and is in gaseous form, and because of the long residence time, transported over long distances. Comprehensive control of mercury emissions therefore remains an important international issue. The crucial steps for designing effective approaches for such control include the quantification of mercury emissions by sources and the identification of geographical characteristics of the emissions. In this study a detailed, high-resolution inventory of Japanese mercury emissions in 2005 was developed to improve understanding of their geographical distribution. Proceeding from a national emissions inventory per source category, emissions were spatially allocated with increasing geographical resolution in a stepwise procedure using statistics from geographic information resources, yielding mercury emissions per prefecture, per municipality and per grid cell of approximately 1 × 1 km. The five prefectures with the highest emissions were Fukuoka, Yamaguchi, Hyogo, Oita, and Hokkaido, accounting for 35.2% of all emissions. In each prefecture a small number of municipalities account for a major share of emissions. Distribution by grid cell is characterized by a concentration of 50% of all emissions in a mere 32 of the 255 954 grid cells over which emissions are distributed in this study. It was also quantitatively confirmed that use of larger grid cells leads to greater uncertainty in emissions distribution. Problems with data collection are clarified and measures to improve the accuracy of future estimation are proposed.

Active intestinal absorption of fluoroquinolone antibacterial agent ciprofloxacin by organic anion transporting polypeptide, Oatp1a5.

Fluoroquinolone antimicrobial drugs are absorbed efficiently after oral administration despite of their hydrophilic nature, implying an involvement of carrier-mediated transport in their membrane transport process. It has been that several fluoroquinolones are substrates of organic anion transporter polypeptides OATP1A2 expressed in human intestine derived Caco-2 cells. In the present study, to clarify the involvement of OATP in intestinal absorption of ciprofloxacin, the contribution of Oatp1a5, which is expressed at the apical membranes of rat enterocytes, to intestinal absorption of ciprofloxacin was investigated in rats. The intestinal membrane permeability of ciprofloxacin was measured by in situ and the vascular perfused closed loop methods. The disappeared and absorbed amount of ciprofloxacin from the intestinal lumen were increased markedly in the presence of 7,8-benzoflavone, a breast cancer resistance protein inhibitor, and ivermectin, a P-glycoprotein inhibitor, while it was decreased significantly in the presence of these inhibitors in combination with naringin, an Oatp1a5 inhibitor. Furthermore, the Oatp1a5-mediated uptake of ciprofloxacin was saturable with a K(m) value of 140 µm, and naringin inhibited the uptake with an IC(50) value of 18 µm by Xenopus oocytes expressing Oatp1a5. Naringin reduced the permeation of ciprofloxacin from the mucosal-to-serosal side, with an IC(50) value of 7.5 µm by the Ussing-type chamber method. The estimated IC(50) values were comparable to that of Oatp1a5. These data suggest that Oatp1a5 is partially responsible for the intestinal absorption of ciprofloxacin. In conclusion, the intestinal absorption of ciprofloxacin could be affected by influx transporters such as Oatp1a5 as well as the efflux transporters such as P-gp and Bcrp.

Contribution of rat pulmonary metabolism to the elimination of lidocaine, midazolam, and nifedipine.

The contribution of the lung to drug metabolism was investigated in rats and the possibility of prediction of in vivo metabolism from in vitro studies using rat pulmonary microsomes was assessed. Lidocaine, midazolam, or nifedipine was administered to rats at a dose of 10 mg/kg by the intra-arterial, intravenous, and intraportal routes. The pulmonary extraction ratios of lidocaine, midazolam, and nifedipine, calculated from the area under the time-plasma concentration curve (AUC) after the intra-arterial and intravenous administrations, were 39.0 +/- 0.5, 18.3 +/- 0.7, and 12.3 +/- 0.3%, respectively. The hepatic extraction ratios of lidocaine, midazolam, and nifedipine, calculated from the AUC after the intraportal and intravenous administrations, were 68.0 +/- 3.3, 52.6 +/- 0.4, and 13.5 +/- 0.2%, respectively. These results showed that both the liver and the lung contributed to the metabolism of these drugs. The above in vivo pulmonary extraction ratios correlated with the in vitro intrinsic clearance values, which were corrected with the protein unbound ratio in microsomes and plasma, suggesting that pulmonary extraction ratios can be predicted quantitatively from in vitro data. The pulmonary intrinsic clearance values of lidocaine, midazolam, and nifedipine in rat microsomes were lower than their hepatic intrinsic clearance, showing that there was an organ difference in metabolism between the liver and lung. Our results support the importance of the estimation of pulmonary metabolism to predict the total clearance more accurately.

A modified loop snare technique for the retrieval of a dislodged central venous catheter.

A dislodged central venous port catheter is typically retrieved using an endovascular approach; however, the retrieval procedure poses a challenge for vascular specialists. The desired outcomes can be successfully achieved with the loop snare technique, an endovascular treatment method for the retrieval of a dislodged central venous port catheter. Herein, we present a case wherein the modified loop snare technique was used for successfully retrieving a dislodged central venous port catheter by reversing the tip of the guidewire inside the right ventricle and advancing it back into the snare.

Treatment of prosthetic vascular graft infection in the groin with ultrasound debridement: A case report.

INTRODUCTION: Prosthetic graft infection (PGI) is associated with low patient survival rates. The effectiveness of ultrasound debridement in chronic wound healing has been previously reported; however, data on the use of ultrasound technology and its effect on the treatment of PGI are still lacking. We report a case in which PGI in the groin was managed by graft removal using ultrasound debridement. PRESENTATION OF CASE: A 70-year-old man was diagnosed with chronic limb-threatening ischemia and underwent a femoral-femoral bypass with a polytetrafluoroethylene graft. Eight months postoperatively, he developed an infection at the femoral incision site. Graft removal was performed using ultrasound debridement. The estimated blood loss was 10 mL. The wound healed, and the patient has remained in good health for 2 years postoperatively. DISCUSSION: When the ultrasonic probe is applied to the wound, ultrasonic energy penetrates into the tissue, and a fibrinolytic action removes necrotic or infected tissue without removing healthy tissue, thereby minimizing bleeding. Using this technique, we were able to perform effective debridement at not only the wound but also the anastomosis. CONCLUSION: It is our opinion that this technique can be used to achieve adequate debridement with little bleeding during graft removal and may provide a new option for the treatment of PGI.

Deep Circumflex Iliac Artery as an Inflow Alternative for Surgical Revascularization: A Case Report.

The common femoral artery (CFA) is the most widely used inflow in all types of surgical revascularization in patients with peripheral artery disease. However, the CFA cannot always be used because of calcification, obstruction, or previous dissection. Here, we report a rare case of selecting the deep circumflex iliac artery (DCIA) as a source of inflow to perform a surgical revascularization in a patient with chronic limb-threatening ischemia. A 62-year-old man was admitted to our hospital due to necrotized third and fifth toes with pain at rest. Computed tomography showed severe stenosis of the CFA, superficial femoral artery, and deep femoral artery, and an entirely stented external iliac artery. The DCIA was identified as the only patent artery. Considering the condition of the other arteries, we selected the DCIA as a source of inflow. Deep circumflex iliac-popliteal bypass was performed with a saphenous vein. The bypass graft was patent 9 months after surgery and limb salvage had been achieved.

Exposure of the cryptic de-adhesive site FNIII14 in fibronectin molecule and its binding to membrane-type eEF1A induce migration and invasion of cancer cells via ß1-integrin inactivation.

Cell migration is a highly coordinated process that involves not only integrin-mediated adhesion but also de-adhesion. We previously found that a cryptic de-adhesive site within fibronectin molecule, termed FNIII14, weakens cell adhesion to the extracellular matrix by inactivating ß1-integrins. Surprisingly, eukaryotic translation elongation factor-1A (eEF1A), an essential factor during protein biosynthesis, was identified as a membrane receptor that mediates the de-adhesive effect of FNIII14. Here, we demonstrate that FNIII14-mediated de-adhesion causes enhanced migration and invasion in two types of highly invasive/metastatic cancer cells, resulting in the initiation of metastasis. Both in vitro migration and invasion of highly invasive human melanoma cell line, Mum2B, were inhibited by a matrix metalloproteinase (MMP)-2/9 inhibitor or a function-blocking antibody against FNIII14 (anti-FNIII14 Ab), suggesting that MMP-mediated exposure of the cryptic de-adhesive site FNIII14 was responsible for Mum2B cell migration and invasion. The MMP-induced FNIII14 exposure was also shown to be functional in the migration and invasion of highly metastatic mouse breast cancer cell line 4T1. Overexpression and knockdown experiments of eEF1A in Mum2B cells revealed that the migration and invasion were dependent on the membrane levels of eEF1A. In vivo experiments using tumor xenograft mouse models derived from Mum2B and 4T1 cell lines showed that the anti-FNIII14 Ab has a significant anti-metastatic effect. Thus, these results provide novel insights into the regulation of cancer cell migration and invasion and suggest promising targets for anti-metastasis strategies.

Three cases of fusion imaging in endovascular treatment of occlusive peripheral artery disease.

Endovascular treatment of peripheral artery disease has dramatically improved in the past decades; however, occlusive or stenotic lesions of the femoral-popliteal artery segment remain a significant challenge for vascular specialists. Real-time guidance based on vessel visualization might be helpful for successful recanalization. Herein, we present three successful cases of fusion imaging during endovascular treatment of the femoral-popliteal artery segments.

Improved intestinal membrane permeability of hexose-quinoline derivatives via the hexose transporter, SGLT1.

Intestinal membrane permeability is an important factor affecting the bioavailability of drugs. As a strategy to improve membrane permeability, membrane transporters are useful targets since essential nutrients are absorbed efficiently via specific transporters. For example, there are reports that intestinal hexose transporters could be used as a tool to improve permeability; however, there has been no direct evidence that the transporter protein, sodium/glucose cotransporter 1 (SGLT1), is involved in the transport of hexose analogs. Accordingly, we examined directly whether the intestinal membrane permeability of hexose analogs can be improved by utilizing SGLT1. Three hexose-quinoline derivatives were synthesized and their interactions with SGLT1 were evaluated. Among the three derivatives, the glucose-quinoline molecule exhibited an inhibitory effect on D-glucose uptake by both rat intestinal brush-border membrane vesicles (BBMVs) and Xenopus oocytes expressing SGLT1. In addition, significant uptake of the glucose-quinoline derivative by Xenopus oocytes expressing SGLT1 was observed by both an electrophysiological assay and direct measurement of the uptake of the compound, while the galactose-quinoline derivative did not show significant uptake via SGLT1. Thus, it was directly demonstrated that SGLT1 could be used as a tool for the improvement of intestinal membrane permeability of drugs by modification to the glucose analogs.

Histological Reactions and the In Vivo Patency Rates of Small Silk Vascular Grafts in a Canine Model.

Objective: To evaluate in vivo patency rates of silk fibroin (SF) vascular grafts and resulting histological reactions in a canine model. Methods: To generate 3.5-mm inner diameter vessels, a combination of plaited silk fibers were wound with cocoon filaments and subsequently coated with an SF solution. The resulting SF grafts (n=35) were implanted into the carotid arteries of male beagles (age, 1-2 years; body weight: 9.0-10.5 kg). Expanded polytetrafluoroethylene (4-mm inner diameter, ePTFE) grafts (n=5) were used as controls. Graft patency was monitored via ultrasonography with histological changes analyzed via microscopic examination. Results: Compared with animals that received the ePTFE grafts, animals that received SF grafts exhibited the same thickness of luminal layers and fibrin accumulation and collagen fiber replacement with endothelialization at 3 months post-implantation via histological examination. The patency rates of the SF and the ePTFE grafts at 6 months post-implantation were 7.8% and 0%, respectively. Conclusion: This canine model study demonstrated that SF grafts induce unique histological reactions but fail to achieve long-term patency.

Rapid endothelialization and thin luminal layers in vascular grafts using silk fibroin.

The histological effects of silk fibroin (SF) in vascular grafts have not been clarified comprehensively in a large-animal model. This study aimed to observe the histological changes in vascular grafts by using Bombyx mori SF in a dog model. A splice graft consisting of SF and control grafts were implanted in the abdominal aorta of dogs, and the histological characteristics of the 2 types of grafts in each splice graft were compared. Five splice grafts consisted of one graft made of polyester (PE) fibers coated with SF and another coated with gelatin, 4 splice grafts consisted of one graft made of SF fibers coated with gelatin and another made of PE fibers coated with gelatin, and 2 splice grafts consisted of one graft made of SF fibers coated with SF and another made of PE fibers coated with SF. The graft made of PE fibers coated with SF showed more endothelial cells than the graft made of PE fibers coated with gelatin. The grafts using SF as a coating material or graft fibers showed a thinner luminal layer than the grafts made of PE fibers coated with gelatin. This study suggests that SF use for vascular grafts has advantages of rapid endothelialization and tendency to form thin luminal layers.

Effectiveness of pirotiodecane, absorption enhancer, on nasal absorption in rabbits.

The absorption enhancing effect of 1-[2-(decylthio) ethyl] azacyclopentan-2-one (Pirotiodecane), on drug permeation across rabbit nasal mucosa was studied. The nasal epithelial mucosa was isolated from rabbit nasal septum and mounted in an Ussing chamber to allow for monitoring of the membrane resistance (Rm), and the permeation of fluorescein isothiocyanate-labeled dextran (FD-4, M.W. 4,400 Da). Treatment with 0.05, 0.1, and 0.2% Pirotiodecane for 60 min decreased Rm, and increased the cumulative amount of FD-4 permeated in a concentration-dependent manner, suggesting that Pirotiodecane possesses passively a disassembly of tight junction to enable the enhanced FD-4 permeation. The remarkable increase in plasma concentration of FD-4 was also observed in intranasal co-administration with 1% Pirotiodecane in rabbits. The Rm was virtually maintained after the removal of Pirotiodecane, although recovery of Rm was not seen. On the other hand, the increase in plasma concentration of FD-4 with intranasal co-administration of 1% Pirotiodecane in rabbits in vivo was not observed in FD-4 administration at 15-60 min after administration of 1% Pirotiodecane alone. It was concluded that Pirotiodecane possesses a relatively short absorption enhancing effect through nasal epithelial.

Long-Term Results of Treatment for Critical Limb Ischemia.

From 2001 to 2012, arterial reconstruction was performed in 306 out of 497 limbs (62%) with critical limb ischemia. The reasons for non-vascularization include high operative risk (36%), extended necrosis or infection (20%), and technical issues (15%). Cumulative patency and limb salvage in collagen disease were significantly worse compared to arteriosclerosis obliterans. Cumulative limb salvage, amputation free survival (AFS), and major adverse limb event and perioperative death (MALE + POD) in patients with end-stage renal disease (ESRD) were significantly worse compared to patients without ESRD, but not significant with regards to graft patency. Our finding suggests that aggressive arterial reconstruction provides satisfactory long-term results in critical limb ischemia so long as case selection for revascularization is properly made. (This article is a translation of J Jpn Coll Angiol 2014; 54: 5-11.).

Factors affecting glucuronidation activity in Caco-2 cells.

Presystemic intestinal metabolism reduces the intestinal absorption and bioavailability of orally administered drugs. The factors affecting glucuronidation activity in Caco-2 cells seeded in Transwell (4.7 cm(2)) require clarification to establish an in-vitro system to assess intestinal glucuronidation metabolism for novel drug development. alpha-Naphthol (alpha-NA), a substrate for UGT1A6 in Caco-2 cells, has often been used as a model substrate for gluruonidation. alpha-Naphthol glucuronidation activity increased from 7 to 21 culture days after seeding in Transwell and stabilized after 21 days. The higher the passage number of Caco-2 cells, the larger the variance of glucuronidation activity, but apical pH did not significantly influence glucuronidation in the pH range of 5.5 to 7.4. When the passage number ranged from 83 to 159, Km,app was highest at passage number 130. In contrast, Vmax,app increased with the passage number. This indicates that the kinetic parameters for glucuronidation in Caco-2 cells are dependent on the passage number of the cells. These results should be useful for establishing the experimental conditions for Caco-2 cells that predict intestinal glucuronidation activity in vivo.