Improvement in ICU Mortality From Sepsis Associated With Recuperation From Septic Multiple-Organ Failure: A Retrospective, Single-Center, Cohort Study.

BACKGROUND: Although mortality due to sepsis has decreased in recent decades, there are few studies on the timing of death during ICU stay. Characteristics of patients related to changes over the years of ICU death and changes in the timing of ICU death will provide new insights for future sepsis management. METHODS: This was a single-center, retrospective study. Patients admitted to the ICU for sepsis between 2005 and 2019 were included in the study. The study period was divided into three five-year intervals, and the timing of death in the ICU was divided into early-stage (1-3 ICU days), mid-stage (4-14 ICU days), and late-stage (15 or more ICU days). Patient characteristics related to ICU death at three five-year intervals and the timing of death were evaluated. RESULTS: ICU mortality for sepsis has decreased over time (2005-2009, 30.2%; 2010-2014, 21.0%; 2015-2019, 12.1%; p<0.01). In the timing of death, only mid-stage mortality decreased. Multiple-organ failure (OR, 4.53; 95% CI, 2.79-7.48) and hematological malignancies (OR, 2.48; 95% CI, 1.19-5.07) were associated with ICU mortality over entire study periods. Only multiple-organ failure was associated with ICU mortality at the five-year intervals (OR, 5.94; 95% CI, 2.73-13.7 for 2005-2009; OR, 4.01; 95% CI, 1.82-9.31 for 2010-2014; OR, 2.58; 95% CI, 1.05-6.59 for 2015-2019). Mid-stage mortality of multiple-organ failure decreased (2005-2009, 12.8%; 2010-2014, 7.6%; 2015-2019, 1.6%; p=0.02). However, early- and late-stage mortality of multiple-organ failure did not change. CONCLUSIONS: Improvement in mid-stage mortality in septic patients with multiple-organ failure can contribute to the improvement of overall ICU mortality in patients with sepsis.

Effect of linezolid on platelet count in critically ill patients with thrombocytopenia.

INTRODUCTION: Linezolid (LZD) is one of the antibiotics used to treat methicillin-resistant Staphylococcus aureus. In Japan, the dose of LZD is not generally adjusted by renal function or therapeutic drug monitoring and is readily available for critically ill patients. The adverse effects of LZD include pancytopenia, especially thrombocytopenia. We investigated the effect of LZD on platelet counts in critically ill patients with thrombocytopenia during admission to the intensive care unit (ICU). METHODS: Fifty-five critically ill patients with existing thrombocytopenia (platelet count < 100 ×103 /µL) who received LZD for five days or more during the period from January 2011 to October 2018 were included. Changes in platelet count and frequency of platelet concentrate (PC) transfusion were evaluated retrospectively. RESULTS: Mean (± standard error) platelet count prior to initiation of LZD was 47 ± 4 ×103 /uL, which increased significantly to 86 ± 13 ×103 /uL on day 15 (p<0.01). Median [interquartile range] duration of LZD therapy was 9 [8-12] days. Thirty-two patients (58.2%) required PC transfusion in the 15-day study period. The daily rate of PC transfusion decreased from 30.2% on days 1-5 to 18.2% on days 11-15. Similar tendencies were observed in patients with non-hematological and hematological disease. CONCLUSION: Thrombocytopenia in critically ill patients in the ICU did not worsen after initiation of LZD therapy, and may be considered for the treatment of MRSA in this setting.

Effect of renal clearance on vancomycin area under the concentration-time curve deviations in critically ill patients.

INTRODUCTION: Augmented renal clearance (ARC) increases vancomycin (VCM) clearance. Therefore, higher VCM doses are recommended in patients with ARC; however, impacts of ARC on the area under the concentration-time curve (AUC) discrepancies between initial dosing design and therapeutic drug monitoring (TDM) period remains unclear. METHODS: We retrospectively collected data from critically ill patients treated with VCM. The primary endpoint was the association between ARC and AUC24-48h deviations. ARC and AUC deviation were defined as a serum creatinine clearance (CCr) ≥130 mL/min/1.73 m2 and an AUC at TDM 30% or more higher than the AUC at the initial dosing design, respectively. The pharmacokinetic profiles of VCM were analyzed with the trough levels or peak/trough levels using the Bayesian estimation software Practical AUC-guided TDM (PAT). RESULTS: Among 141 patients (median [IQR]; 66 [58-74] years old; 30% women), 35 (25%) had ARC. AUC deviations were significantly more frequent in the ARC group than in the non-ARC group (20/35 [57.1%] and 17/106 [16.0%] patients, respectively, p < 0.001). Age- and sex-adjusted multivariate analyses revealed that the number of VCM doses before TDM ≥5 (odds ratio, 2.56; 95% confidence interval [CI]: 1.01-6.44, p = 0.047) and CCr ≥130 mL/min/1.73 m2 were significantly associated with AUC deviations (odds ratio, 7.86; 95%CI: 2.91-21.19, p < 0.001). CONCLUSION: Our study clarifies that the AUC of VCM in patients with ARC is higher at the time of TDM than at the time of dosage design.

Albumin protects the ultrastructure of the endothelial glycocalyx of coronary arteries in myocardial ischemia-reperfusion injury in vivo.

Myocardial ischemia-reperfusion (I/R) injury induces endothelial glycocalyx (GCX) degradation. Several candidate GCX-protective factors including albumin have been identified, few have been demonstrated in in vivo studies and most albumins used to date have been heterologous. Albumin is a carrier protein for sphingosine 1-phosphate (S1P), which has protective effects on the cardiovascular system. However, changes inhibited by albumin in the endothelial GCX structure in I/R in vivo via the S1P receptor has not been reported. In this study, we aimed to determine whether albumin prevents the shedding of endothelial GCX in response to I/R in vivo. Rats were divided into four groups: control (CON), I/R, I/R with albumin preload (I/R + ALB), and I/R + ALB with S1P receptor agonist fingolimod (I/R + ALB + FIN). FIN acts as an initial agonist of S1P receptor 1 and downregulates the receptor in an inhibitory manner. The CON and I/R groups received saline and I/R + ALB and I/R + ALB + FIN groups received albumin solution before left anterior descending coronary artery ligation. Our study used rat albumin. Shedding of endothelial GCX was evaluated in the myocardium by electron microscopy, and the concentration of serum syndecan-1 was measured. Thus, albumin administration maintained the structure of endothelial GCX and prevented shedding of endothelial GCX via the S1P receptor in myocardial I/R, and FIN annihilated the protective effect of albumin against I/R injury.

Optimal target blood pressure in elderly with septic shock (OPTPRESS) trial: study protocol for a randomized controlled trial.

BACKGROUND: Hemodynamic stabilization is a core component in the resuscitation of septic shock. However, the optimal target blood pressure remains debatable. Previous randomized controlled trials suggested that uniformly adopting a target mean arterial pressure (MAP) higher than 65 mmHg for all adult septic shock patients would not be beneficial; however, it has also been proposed that higher target MAP may be beneficial for elderly patients, especially those with arteriosclerosis. METHODS: A multicenter, pragmatic single-blind randomized controlled trial will be conducted to compare target MAP of 80-85 mmHg (high-target) and 65-70 mmHg (control) in the resuscitation of septic shock patients admitted to 28 hospitals in Japan. Patients with septic shock aged ≥65 years are randomly assigned to the high-target or control groups. The target MAP shall be maintained for 72 h after randomization or until vasopressors are no longer needed to improve patients' condition. To minimize the adverse effects related to catecholamines, if norepinephrine dose of ≥ 0.1 µg/kg/min is needed to maintain the target MAP, vasopressin will be initiated. Other therapeutic approaches, including fluid administration, hydrocortisone use, and antibiotic choice, will be determined by the physician in charge based on the latest clinical guidelines. The primary outcome is all-cause mortality at 90 days after randomization. DISCUSSION: The result of this trial will provide great insight on the resuscitation strategy for septic shock in the era of global aged society. Also, it will provide the better understanding on the importance of individualized treatment strategy in hemodynamic management in critically ill patients. TRIAL REGISTRATION: UMIN Clinical Trials Registry; UMIN000041775. Registered 13 September 2020.

Frequent tracheal suctioning is associated with extubation failure in patients with successful spontaneous breathing trial: a single-center retrospective cohort study.

BACKGROUND: Extubation failure, i.e., reintubation in ventilated patients, is a well-known risk factor for mortality and prolonged stay in the intensive care unit (ICU). Although sputum volume is a risk factor, the frequency of tracheal suctioning has not been validated as a predictor of reintubation. We conducted this study to examine whether frequent tracheal suctioning is a risk factor for reintubation. PATIENTS AND METHODS: We included adult patients who were intubated for > 72 h in the ICU and extubated after completion of spontaneous breathing trial (SBT). We compared the characteristics and weaning-related variables, including the frequency of tracheal suctioning between patients who required reintubation within 24 h after extubation and those who did not, and examined the factors responsible for reintubation. RESULTS: Of the 400 patients enrolled, reintubation was required in 51 (12.8%). The most common cause of reintubation was difficulty in sputum excretion (66.7%). There were significant differences in sex, proportion of patients with chronic kidney disease, pneumonia, ICU admission type, the length of mechanical ventilation, and ICU stay between patients requiring reintubation and those who did not. Multivariate analysis showed frequent tracheal suction (> once every 2 h) and the length of mechanical ventilation were independent factors for predicting reintubation. CONCLUSION: We should examine the frequency of tracheal suctioning > once every 2 h in addition to the length of mechanical ventilation before deciding to extubate after completion of SBT in patients intubated for > 72 h in the ICU.

Comparison of the National Early Warning Scores and Rapid Emergency Medicine Scores with the APACHE II Scores as a Prediction of Mortality in Patients with Medical Emergency Team Activation: a Single-centre Retrospective Cohort Study.

INTRODUCTION: The medical emergency team enables the limitation of patients' progression to critical illness in the general ward. The early warning scoring system (EWS) is one of the criteria for medical emergency team activation; however, it is not a valid criterion to predict the prognosis of patients with MET activation. AIM: In this study, the National Early Warning Score (NEWS) and Rapid Emergency Medicine Score (REMS) was compared with that of the Acute Physiology and Chronic Health Evaluation II (APACHE II) score in predicting the prognosis of patients who had been treated a medical emergency team. MATERIAL AND METHODS: In this single-centre retrospective cohort study, patients treated by a medical emergency team between April 2013 and March 2019 and the 28-day prognosis of MET-activated patients were assessed using APACHE II, NEWS, and REMS. RESULTS: Of the 196 patients enrolled, 152 (77.5%) were men, and 44 (22.5%) were women. Their median age was 68 years (interquartile range: 57-76 years). The most common cause of medical emergency team activation was respiratory failure (43.4%). Univariate analysis showed that APACHE II score, NEWS, and REMS were associated with 28-day prognostic mortality. There was no significant difference in the area under the receiver operating characteristic curve of APACHE II (0.76), NEWS (0.67), and REMS (0.70); however, the sensitivity of NEWS (0.70) was superior to that of REMS (0.47). CONCLUSION: NEWS is a more sensitive screening tool like APACHE II than REMS for predicting the prognosis of patients with medical emergency team activation. However, because the accuracy of NEWS was not sufficient compared with that of APACHE II score, it is necessary to develop a screening tool with higher sensitivity and accuracy that can be easily calculated at the bedside in the general ward.

Using an ICU Diary to Communicate With Family Members of COVID-19 Patients in ICU: A Case Report.

Visitation restrictions for family members are problematic in intensive care management due to the COVID-19 pandemic. We analyzed the usefulness of an intensive care unit (ICU) diary about the experiences of family members of critical COVID-19 patients. Four family members of 2 COVID-19 patients participated in this report. Both patients were transferred to our ICU after 2 weeks of treatment at another ICU. An ICU diary was given to their family members post-transfer. The family members were interviewed before and after the patients' discharge; the recorded interviews were analyzed and categorized into several clusters using a text mining method. Five categories regarding their anxious feelings were classified before the use of the ICU diary, and 3 categories were based on their positive feelings after the use of the ICU diary. Intensive care unit diaries may be beneficial for disclosing patients' information when visitation restrictions are exercised due to the COVID-19 pandemic.

Cardiac Failure Requiring Veno-Arterial Extracorporeal Membrane Oxygenation (VA-ECMO) Management in a Refeeding Syndrome Patient with Diabetic Ketoacidosis: A Case Report.

BACKGROUND Refeeding syndrome is a complex metabolic disorder that develops following rapid nutritional administration after a long period of undernutrition. The onset mechanism involves intracellular transport of phosphorus, potassium, and water, in association with rapid glucose administration. The resulting hypophosphatemia is extremely dangerous and can cause severe heart failure and fatal arrhythmia. We successfully used extracorporeal cardiopulmonary support to manage a case of refeeding syndrome that occurred during the course of treatment of diabetic ketoacidosis. There are only a few reports of the use of cardiopulmonary support for the treatment of refeeding syndrome. CASE REPORT A 72-year-old man was admitted to the hospital for treatment of diabetic ketoacidosis. Despite receiving insulin and nutrition therapy, QT prolongation and ventricular fibrillation appeared on the electrocardiogram. Although coronary angiography was performed in consideration of the possibility of ischemic heart disease, no significant stenosis of the coronary arteries was identified. Due to persistent hypotension and recurrence of ventricular fibrillation, extracorporeal cardiopulmonary support was commenced in the ICU. His serum phosphorus level showed a marked decrease on his first day in the ICU, for which daily replacement therapy was administered during his ICU stay. No fatal arrhythmia developed thereafter. He was weaned off extracorporeal cardiopulmonary support on the fourth day of his ICU stay and was subsequently discharged from the hospital. CONCLUSIONS We suggest vigilant monitoring of electrolytes, including phosphate levels, in diabetic ketoacidosis patients, and active circulatory support, as required, in patients with refeeding syndrome.

Validation of the relationship between coagulopathy and localization of hydroxyethyl starch on the vascular endothelium in a rat hemodilution model.

Various anticoagulant properties have been associated with hydroxyethyl starch (HES). However, the mechanism remains unclear and it has not been fully considered whether these properties are beyond the dilutional effect itself. The aim of this study was to reproduce the coagulopathy induced by HES and to test the hypothesis that the coagulopathy is caused by endothelial or glycocalyx damage due to localization of HES on the endothelium, which is caused by the high shear viscosity of dilutional blood. Using a rat model, we compared blood coagulability measured by Sonoclot, levels of endothelial and glycocalyx damage markers and coagulation factors, and blood shear viscosity when hemodilution was performed with physiological saline (PS), 6% HES 130/0.4 in PS, and 10% HES 200/0.5 in PS. We also evaluated the localization rates of fluorescently labeled HES on endothelium in the isolated aorta. HES decreased the fibrin gel formation rate more than did PS. HES was shown to cover the endothelium, possibly due to its high shear viscosity, and this mechanism potentially acted to protect, rather than damage, the endothelium and glycocalyx. However, this covering effect may be the cause of coagulopathy due to inhibition of von Willebrand factor secretion from the endothelium.

Effects of anesthetic agents on contractions of the pregnant rat myometrium in vivo and in vitro.

BACKGROUND: Several anesthetic agents are used in cesarean sections for both regional and general anesthesia purposes. However, there are no data comparing the in vivo effects of propofol, sevoflurane, and dexmedetomidine on the contraction of the myometrium in pregnant rats. The aim of this study was to investigate the effect of these anesthetic agents on myometrial contraction and elucidate the underlying mechanisms. METHODS: Contraction force and frequency changes in response to propofol, dexmedetomidine, or sevoflurane were evaluated in vivo and in vitro. To test the effect of arachidonic acid on myometrial contraction enhanced by dexmedetomidine, changes in myometrial contraction with dexmedetomidine after administration of indomethacin were evaluated. The amount of phosphorylated myosin phosphatase target subunit 1 (MYPT1) in the membrane fraction was expressed as a percentage of the total fraction by Western blot analysis. RESULTS: This study demonstrated that dexmedetomidine enhances oxytocin-induced contraction in the myometrium of pregnant rats, whereas propofol and sevoflurane attenuate these contractions. The dexmedetomidine-induced enhancement of myometrial contraction force was abolished by the administration of indomethacin. Propofol did not affect oxytocin-induced MYPT1 phosphorylation, whereas sevoflurane attenuated oxytocin-induced MYPT1 phosphorylation. CONCLUSIONS: Inhibition of myofilament calcium sensitivity may underlie the inhibition of myometrial contraction induced by sevoflurane. Arachidonic acid may play an important role in the enhancement of myometrial contraction induced by dexmedetomidine by increasing myofilament calcium sensitivity. Dexmedetomidine may be used as a sedative agent to promote uterine muscle contraction and suppress bleeding after fetal delivery.

Endoscopic Insertion of Nasojejunal Feeding Tube at Bedside for Critically Ill Patients: Relationship between Tube Position and Intragastric Countercurrent of Contrast Medium.

Background and Oblectives: We evaluated the success rate of endoscopically positioned nasojejunal feeding tubes and the intragastric countercurrent of contrast medium thereafter. METHOD: This retrospective observational study investigated patients who were admitted to a single intensive care unit and required endoscopic placement of a post-pyloric feeding tube between January 2010 and June 2016. The feeding tube was grasped with forceps via a transoral endoscope and inserted into the duodenum or jejunum. Thereafter, we assessed the position of the tube and the intragastric countercurrent using abdominal radiography with contrast medium. RESULTS: The tube tip was inserted at the jejunum and the duodenal fourth portion in 55.8 and 33.6% of patients, respectively. The tip of the inserted tube had moved into the jejunum of 71.7% of patients by the following day. The countercurrent rate was significantly lower among patients with a tube inserted into the duodenal fourth portion or more distal than among those with tubes inserted more proximally (8.4 vs. 45.4%, p = 0.0022). CONCLUSIONS: The endoscopic insertion and positioning of a nasojejunal feeding tube seemed effective because the rate of tube insertion into the duodenal fourth portion or more distal was about 90%. The findings of intragastric countercurrents indicated that feeding tubes should be inserted into the duodenal fourth portion or beyond to prevent vomiting and the aspiration of enteral nutrients.

Sevoflurane Promotes Regeneration of the Endothelial Glycocalyx by Upregulating Sialyltransferase.

BACKGROUND: During ischemia-reperfusion injury, the endothelial glycocalyx is damaged by oxidative stress-induced release of hydrogen peroxide, leading to decreased endothelium-dependent vasodilation. The regenerative effects of sevoflurane on the endothelial glycocalyx and endothelium-dependent vasodilation in oxidative stress remain unclear. Sialic acid, which is a component of the glycocalyx, plays a key role in antioxidant activity and is catalyzed by the sialyltransferase, ST6Gal-I. We hypothesized that ST6Gal-I is involved in the sevoflurane-induced promotion of endothelial glycocalyx restoration and endothelium-dependent vasodilation after oxidative stress. MATERIALS AND METHODS: To assess vasodilation, isometric tension in the rat aorta was measured. Aortic rings were treated with 0.5 mM hydrogen peroxide pre-exposure or post exposure to sevoflurane, with or without an ST6Gal-I inhibitor. The rings were then used for glycocalyx imaging using fluorescein isothiocyanate-labeled lectin staining and for immunohistochemistry for ST6Gal-I. RESULTS: Vasodilation was significantly decreased by treatment with hydrogen peroxide compared to controls. Application of sevoflurane after treatment with hydrogen peroxide revived endothelium-dependent vasodilatation, whereas pretreatment with sevoflurane had no such effect. Sevoflurane after-treatment revived the intensity of fluorescence of the endothelial glycocalyx compared to the hydrogen peroxide group. However, pretreatment with sevoflurane had no such effect. Sevoflurane significantly upregulated the reduced expression of ST6Gal-I induced by hydrogen peroxide treatment. CONCLUSIONS: Sevoflurane exerts regenerative effects on endothelium-dependent vasodilation and the endothelial glycocalyx following oxidative stress in the rat aorta.

Comparative Study of TOF-Cuff, a New Neuromuscular Blockade Monitor, and TOF-Watch, an Acceleromyography.

Train-of-four ratios were recorded to assess the agreement between the TOF-Cuff and TOF-Watch, and residual paresis was assessed to evaluate the clinical utility of TOF-Cuff. Train-of-four ratios were evaluated using Lin concordance correlation coefficient and Bland-Altman analyses. Measured train-of-four ratios demonstrated high accuracy and precision over the entire range of train-of-four ratios. Although precision and Lin concordance correlation coefficients decreased with train-of-four ratios >0.7, none of the patients showed signs of residual paresis. Because TOF-Cuff underestimated train-of-four ratios in the recovery period, the clinical safety of train-of-four ratios >0.9 indicated by TOF-Cuff is unclear; the issue of residual paresis requires future research that rigorously evaluates outcomes.

Desflurane inhibits endothelium-dependent vasodilation more than sevoflurane with inhibition of endothelial nitric oxide synthase by different mechanisms.

The effects of desflurane on endothelium-dependent vasodilation remain uncertain, whereas sevoflurane is known to inhibit it. Endothelium-dependent vasodilation is mainly mediated by endothelial nitric oxide synthase. The effects of desflurane on endothelium-dependent vasodilation were compared with those of sevoflurane, and inhibition mechanisms, including phosphorylation of endothelial nitric oxide synthase and the calcium pathway, were evaluated for the two anesthetics. We hypothesized that desflurane would inhibit endothelium-dependent vasodilation in a concentration-dependent manner more than sevoflurane, with inhibition of a calcium pathway. Isolated rat aortic rings were randomly assigned to treatment with desflurane or sevoflurane for measurements of the vasodilation ratio. To determine NO production with desflurane and sevoflurane, an in vitro assay was performed with cultured bovine aortic endothelial cells. These cells were also used for measurement of intracellular calcium or Western blotting. For endothelium-dependent vasodilation, the ratio of vasodilation was more significantly inhibited by 11.4% desflurane than by 4.8% sevoflurane. Inhibition did not between 5.7% desflurane and 2.4% sevoflurane. No inhibitory effect of desflurane or sevoflurane was observed in endothelium-denuded aorta. Desflurane inhibited nitric oxide production caused by stimulation of bradykinin significantly more than sevoflurane. Desflurane had a greater suppressive effect on the bradykinin-induced increase in intracellular calcium concentration than did sevoflurane. Sevoflurane, but not desflurane, inhibited phosphorylation of the serine 1177 residue by bradykinin stimulation. Desflurane inhibited endothelium-dependent vasodilation more than sevoflurane through inhibition of a calcium pathway. Sevoflurane inhibited endothelium-dependent vasodilation by inhibition of phosphorylation of the serine 1177 residue of endothelial nitric oxide synthase.

Recovery of postoperative cognitive function in elderly patients after a long duration of desflurane anesthesia: a pilot study.

Postoperative cognitive dysfunction (POCD) increases morbidity and mortality. The mechanisms underlying POCD remain elusive; however, systemic responses induced by anesthesia and surgery might trigger neuroinflammation and POCD. Desflurane is a preferable volatile anesthetic agent for elderly patients because it facilitates shorter recovery from general anesthesia. The aim of this study was to determine whether quality of emergence and cognitive function in elderly patients undergoing a long duration desflurane anesthesia are better than those in the case of sevoflurane anesthesia. Forty-two patients who were older than 65 years of age and scheduled for surgery of more than 4 h in duration were enrolled in this study. Patients were randomly assigned to a desflurane anesthesia group (D group) and sevoflurane anesthesia group (S group). General anesthesia was maintained with 3.5 % desflurane (D group) and 1.0 % sevoflurane (S group). The Mini-Mental State Examination (MMSE) was used for assessing cognitive function 24 h before and after surgery. Postoperative MMSE score in the D group was significantly improved compared to that in the preoperative period. In conclusion, elderly patients undergoing desflurane anesthesia have significantly better quality of emergence and may have better cognitive function than those in elderly patients undergoing sevoflurane anesthesia.

Novel substrates for nitric oxide synthases.

Enzymatic generation of nitric oxide (NO) by nitric oxide synthase (NOS) consists of two oxidation steps. The first step converts L-arginine to N(G)-hydroxy-L-arginine (NOHA), a key intermediate, and the second step converts NOHA to NO and L-citrulline. To fully probe the substrate specificity of the second enzymatic step, an extensive structural screening was carried out using a series of N-alkyl (and N-aryl) substituted-N'-hydroxyguanidines (1-14). Among the eleven N-alkyl-N'-hydroxyguanidines evaluated, N-n-propyl (2), N-iso-propyl (3), N-n-butyl (4), N-s-butyl (5), N-iso-butyl (6), N-pentyl (8) and N-iso-pentyl (9) derivatives were efficiently oxidized by the three isoenzymes of NOS (nNOS, iNOS and eNOS) to generate NO. N-Butyl-N'-hydroxyguanidine (4) was the best substrate for iNOS (K(m)=33 microM) and N-iso-propyl-N'-hydroxyguanidine (3) was the best substrate for nNOS (K(m)=56 microM). When the alkyl substituents were too small (such as ethyl 1) or too large (such as hexyl 10 and cyclohexyl 11), the activity decreased significantly. This suggests that the van der Waals interaction between the alkyl group and the hydrophobic cavity in the NOS active site contributes significantly to the relative reactivity of compounds 3-11. Moreover, five N-aryl-N'-hydroxyguanidines were found to be good substrates for iNOS, but not substrates for eNOS and nNOS. N-phenyl-N'-hydroxyguanidine was the best substrate among them (K(m)=243 microM). This work demonstrates that N-alkyl substituted hydroxyguanidine compounds are novel NOS substrates which 'short-circuit' the first oxidation step of NOS, and N-aryl substituted hydroxyguanidine compounds are isoform selective NOS substrate.