Snakebite is a neglected tropical disease of global importance affecting at least 2.5 million people with more than 100,000 deaths annually.1Harrison R.A. Hargreaves A. Wagstaff S.C. et al.Snake envenoming: a disease of poverty.PLoS Negl Trop Dis. 2009; 3: e569Crossref PubMed Scopus (288) Google Scholar, 2World Health Organization Prevalence of snakebite envenoming. 2017.https://www.who.int/snakebites/epidemiology/en/Google Scholar Morbidity and mortality are high in countries such as Myanmar, where recent hospital data reported 15,000 to 20,000 cases per year with case-fatality ratio of 10.9%.3Myo-Khin Theingi-Nyunt Nyan-Tun-Oo et al.Prognostic indicators in patients with snakebite: analysis of two-year data from a township hospital in central Myanmar.WHO South East Asia J Public Health. 2012; 1: 144-150Crossref PubMed Google Scholar Experience elsewhere suggests that hospital-based data may underestimate the actual burden of snakebite by more than two-thirds.4Fox S. Rathuwithana A.C. Kasturiratne A. et al.Underestimation of snakebite mortality by hospital statistics in the Monaragala District of Sri Lanka.Trans R Soc Trop Med Hyg. 2006; 100: 693-695Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 5Mohapatra B. Warrell D.A. Suraweera W. et al.Snakebite mortality in India: a nationally representative mortality survey.PLoS Negl Trop Dis. 2011; 5e1018Crossref PubMed Scopus (318) Google Scholar To assess outcomes of snakebite cases at Mandalay General Hospital, we established a clinical data collection system. This major hospital serves as a regional referral center for snakebite. In this region of Myanmar, Eastern Russell’s Viper (ERV; Daboia siamensis) snakebite is of the utmost importance given the high incidence of acute kidney injury (AKI) following envenoming.6Myint-Lwin Warrell D.A. Phillips R.E. et al.Bites by Russell's viper (Vipera russelli siamensis) in Burma: haemostatic, vascular, and renal disturbances and response to treatment.Lancet. 1985; 2: 1259-1264Abstract PubMed Scopus (126) Google Scholar, 7Warrell D.A. Snake venoms in science and clinical medicine. 1. Russell's viper: biology, venom and treatment of bites.Trans R Soc Trop Med Hyg. 1989; 83: 732-740Abstract Full Text PDF PubMed Scopus (181) Google Scholar The primary purpose of this clinical audit, which represents one arm of an Australian Department of Foreign Affairs and Trade–funded foreign aid project to improve the outcomes of snakebite patients in Myanmar,8White J. Mahmood M.A. Alfred S. et al.A comprehensive approach to managing a neglected, neglected tropical disease: The Myanmar Snakebite Project (MSP).Toxicon X. 2019; 1: 100001Crossref Scopus (5) Google Scholar is to provide accurate information to local health authorities to improve health care policies and resource allocation. In addition, we wanted to examine the clinical variables that affect the development of AKI following ERV envenoming. We report 12 months of observational data pertaining to ERV snakebites. A total of 965 patients presented to Mandalay General Hospital after snakebites during the 12-month period. Data for 17 patients were incomplete, leaving 948 for analysis. Bites were attributed to ERV in 686 cases (72.4%), cobra (Naja kaouthia and Naja mandalayensis) in 17 (1.8%), “green snake” (Trimeresurus albolabris) in 61 (6.4%), krait (Bungarus spp.) in 4 (0.4%), other snakes including nonvenomous species in 35 (3.7%), and unknown snakes in 145 (15.3%). In most cases, the dead snake was brought to the hospital and identified by medical staff. In the others, the diagnostic clinical syndrome combined with recognition by the patient of the familiar "mwe bwe" (ERV, Daboia siamensis) was accepted as sufficient identification. This report concentrates on ERV cases given that envenoming from this species alone accounts for 70% of all patients requiring acute nephrological care in Myanmar.9Mon Hla Patterns of acute renal failure in Burma.in: Oxford Textbook of Medicine. Second Edition. Vol 2. 18. Oxford University Press, Oxford, UK1987: 179Google Scholar Patients were typically male (64.9%) and had been bitten on the lower limbs during farm work. Median age was 34 (interquartile range [IQR] 24). Appropriate first aid (pressure pad and immobilization) was rarely applied. Tight tourniquets were applied commonly (77.8%); other interventions included incision (5.8%) and tattooing (8.9%). The first point of health care contact for most was either a rural health center or township hospital (82.8%), and traditional healers were consulted first in 13.9%. The median time from bite to arrival at a health care facility was 1.5 hours (IQR 2.19); the median time from bite to administration of the first dose of antivenom was 2 hours (IQR 3.5). Almost all patients received antivenom (679, 98.9%); 295 cases (43%) received treatment considered compliant with national guidelines (initial dose of 8 vials of Burma Pharmaceutical Industry ERV monovalent antivenom, - F[ab']2 fragments of equine hyperimmune plasma, for patients with significant features of ERV envenoming, see Figure 1). A common but noncompliant pattern in the remaining cases involved 1 to 2 vials given at a small health care facility followed by transfer to a larger facility where more antivenom was given. In this study, AKI was defined pragmatically as a composite endpoint of either requirement for dialysis or, in the absence of requirement for dialysis, a peak serum creatinine level of >120 μmol/l in men or >100 μmol/l in women and a pattern of rising serial creatinine consistent with AKI. The clinical consequences of envenoming are listed in Table 1. AKI was extremely common, manifesting in 488 patients (71% of entire cohort). Of these 488, dialysis (predominantly haemodialysis) was required in 213 (31% of entire cohort), whereas the other 275 patients (40% of entire cohort) suffered a pathological rise in serum creatinine but did not need dialysis (median peak serum creatinine 245.5 μmol/l [IQR 332] in male patients, 260.5 μmol/l [IQR 322] in female patients). Female patients were 1.8 times more likely than male patients to develop AKI (P < 0.01). AKI developed more frequently in older patients, with odds ratio (OR) of 5.5 (11.4 for survivors) in those >64 years compared with those <15 years (P < 0.01).Table 1Clinical features of 686 cases of Russell’s Viper envenomingClinical featuresNumber (% of 686)AKI group (% of 488)No-AKI group (% of 198)Acute kidney injury488 (71)Coagulopathy465 (67)373 (76)92 (47)Thrombocytopenia461 (67)414 (85)47 (24)Capillary leak240 (35)216 (44)24 (12) Pulmonary edema16142 Periorbital edema11810612 Conjunctival edema91829 Generalized edema15141Shock103 (15)92 (19)11 (6)Bite site infection74 (11)51 (11)23 (12)Local necrosis44 (6.4)33 (7)11 (6)Gastrointestinal bleeding38 (5.5)33 (7)5 (3)Septicemia29 (4.2)26 (5)3 (2)Panhypopituitism19 (2.7)19 (4)0Ophthalmoplegia2 (0.29)2 (0.4)0None59 (8.6)In this study, AKI was defined pragmatically as a composite endpoint of either requirement for dialysis or, in the absence of requirement for dialysis, a peak serum creatinine level of >120 μmol/l in men or >100 μmol/l in women and a pattern of rising serial creatinine consistent with AKI. Open table in a new tab In this study, AKI was defined pragmatically as a composite endpoint of either requirement for dialysis or, in the absence of requirement for dialysis, a peak serum creatinine level of >120 μmol/l in men or >100 μmol/l in women and a pattern of rising serial creatinine consistent with AKI. Multivariate analysis (Table 2) showed that the time interval from bite to antivenom administration (irrespective of the initial dosage of antivenom) was the strongest predictor of subsequent AKI (OR 1.7 when antivenom was given at 1–2 hours compared with 0–1 hour, P < 0.05; OR 3.2 at 2–3 hours compared with 0–1 hour, P < 0.01; OR 4.2 at 3–4 hours compared with 0–1 hour, P < 0.01; OR 12.4 at 4–5 hours compared with 0–1 hour, P < 0.01). This effect was observed across the 2 AKI subgroups as defined by dialysis requirement or serum creatinine rise without need for dialysis. Early administration of antivenom was also associated with shorter duration of coagulopathy (for patients receiving antivenom at 10 hours compared with those at 0–1 hour, P < 0.001).Table 2Significant explanatory variables affecting AKI as determined by multivariate logistic regressionExplanatory variablesGroupAKIa (surviving patients only in italics)Sig. cf.Ref.GroupOdds ratioLower 95% CIUpper 95% CIAge groupb (cf. 0–15 yr)50–64 yrP < 0.05P < 0.052.83.01.11.17.28.3Age group (cf. 0–15 yr)>64 yrP < 0.01P < 0.015.511.41.62.519.651.5Gender (cf. M)FP < 0.01P < 0.021.82.01.21.32.73.0Time bite to first AVc (cf. 0–1 h)1–2 hP < 0.05P = 0.0551.71.81.01.03.03.2Time bite to first AV (cf. 0–1 h)2–3 hP < 0.01P < 0.013.22.81.51.36.86.2Time bite to first AV (cf. 0–1 h)3–4 hP < 0.01P < 0.014.24.21.61.511.111.6Time bite to first AV (cf. 0–1 h)4–5 hP < 0.01P < 0.0112.412.52.52.362.967.3Time bite to first HCFd (cf. 0–1 h)4–5 hP = 0.055P < 0.059.810.01.11.189.491.5Time bite to first HCF (cf. 0–1 h)>10 hP < 0.02P < 0.024.75.21.41.416.019.7AKI, acute kidney injury; AV, antivenom; cf., compared with; CI, confidence interval; F, female; HCF, health care facility; M, male; Sig.cf.Ref.Group, significance compared with reference group.Dependent variables:aAKI, as defined as a composite endpoint of either requirement for dialysis or, in the absence of requirement for dialysis, a peak serum creatinine level of >120 μmol/l in men or >100 μmol/l in women and a pattern of rising serial creatinine consistent with AKI.Categorical variables entered into the model, derived by coding continuous explanatory variables that did not exhibit a normal distribution:bAge group, years: 0–15 (ref.); 16–19; 20–29; 30–49; 50–64; >64.cTime from bite to first antivenom administration, hours: 0–1 (ref.); 1–2; 2–3; 3–4; 4–5; 5–6; 6–10; >10.dTime from bite to arrival at first HCF. Open table in a new tab AKI, acute kidney injury; AV, antivenom; cf., compared with; CI, confidence interval; F, female; HCF, health care facility; M, male; Sig.cf.Ref.Group, significance compared with reference group. Dependent variables:aAKI, as defined as a composite endpoint of either requirement for dialysis or, in the absence of requirement for dialysis, a peak serum creatinine level of >120 μmol/l in men or >100 μmol/l in women and a pattern of rising serial creatinine consistent with AKI. Categorical variables entered into the model, derived by coding continuous explanatory variables that did not exhibit a normal distribution:bAge group, years: 0–15 (ref.); 16–19; 20–29; 30–49; 50–64; >64.cTime from bite to first antivenom administration, hours: 0–1 (ref.); 1–2; 2–3; 3–4; 4–5; 5–6; 6–10; >10.dTime from bite to arrival at first HCF. The development of AKI was an important clinical event given that AKI was associated significantly with mortality. The overall mortality was 12.2% (84 of 686) among the entire cohort of 686 ERV cases. More specifically, mortality was 20.2% (43 of 213) in those who required dialysis compared with 10.2% (28 of 275) in those with AKI but did not require dialysis (P = 0.002), and 6.6% (13 of 198) in those who did not develop AKI (P < 0.001). This study reveals the devastating scourge of snakebites in Myanmar. It highlights significant morbidity and mortality from ERV envenoming. The high rate of AKI (71%) was observed in a tertiary hospital caring for severely envenomed patients. The true rate of AKI consequent to all ERV bites may be lower, as not all patients require transfer to a tertiary hospital. Calculating the true risk of AKI requires accurate knowledge of snakebite incidence in the community. Our community-based survey of 2 rural townships in Mandalay indicated that the true incidence of snakebite in Myanmar may be twice as high as that derived from hospital data.S1 Evidently, a nationwide survey of all levels of the health care system is required. Our finding that female patients were 1.8 times more likely than male patients to develop AKI after ERV envenoming warrants further investigation. Factors such as smaller body mass relative to venom load, nutritional status, pregnancy, and anemia may contribute to this gender disparity. The pathogenesis of AKI after ERV envenoming is incompletely known, but it is likely to be multifactorial, including microvascular fibrin deposition,S2 direct nephrotoxicity,S3 and hypotension.6Myint-Lwin Warrell D.A. Phillips R.E. et al.Bites by Russell's viper (Vipera russelli siamensis) in Burma: haemostatic, vascular, and renal disturbances and response to treatment.Lancet. 1985; 2: 1259-1264Abstract PubMed Scopus (126) Google Scholar Until more effective therapies become available, antivenom will remain the mainstay of treatment. Our finding that a shorter delay before antivenom had a better outcome is in broad agreement with 2 other reports based on smaller cohorts of patients.3Myo-Khin Theingi-Nyunt Nyan-Tun-Oo et al.Prognostic indicators in patients with snakebite: analysis of two-year data from a township hospital in central Myanmar.WHO South East Asia J Public Health. 2012; 1: 144-150Crossref PubMed Google Scholar,S4 Over the past 4 years, Australian, UK, and Myanmar colleagues have helped Myanmar become self-sufficient in antivenom production8White J. Mahmood M.A. Alfred S. et al.A comprehensive approach to managing a neglected, neglected tropical disease: The Myanmar Snakebite Project (MSP).Toxicon X. 2019; 1: 100001Crossref Scopus (5) Google Scholar; however, increasing the production of antivenom may not be enough to improve clinical outcomes. In response to our finding of an association between time to antivenom and AKI, the Myanmar Ministry of Health is reviewing its policies about distributing more antivenom to rural health care centers and township hospitals that are within closer reach of snakebite patients. A limitation of this study is the lack of independent identification of snakes; the ERV cohort was based on assumed snake identity. Venom detection testing was not available, and very few dead snakes brought in by patients were kept for identification, although those that were available were predominantly ERVs. This limitation reflects the realities of clinical practice, where experienced clinicians must make pragmatic decisions about the likely culprit snake. In Mandalay Division of Myanmar, snakebite patients presenting with incoagulable blood are most likely to have ERV envenoming. The only other snakes causing this effect are green pit vipers (genus Trimeresurus), whose envenoming is unresponsive to ERV antivenom, and only very rarely results in AKI. Although we had observed a beneficial effect of shorter time to antivenom, administration of 8 vials of antivenom compared with fewer than 8 vials did not correlate with decreased likelihood of AKI on either univariate or multivariate analysis. In this regard, several points are worth considering. First, this was an observational study, not a controlled clinical trial. Confounding factors, such as antivenom availability and clinical bias, may have influenced the initial antivenom dose. Antivenom rationing was common in rural health facilities; it was likely that higher antivenom dose was reserved for patients judged to have severe envenoming. Second, antivenom-specific factors such as unreliable storage cold chain and variable neutralizing potency may have limited its clinical efficacy. Efforts are under way to address these concerns and to determine the optimal initial antivenom dose through controlled clinical trials. All the authors declared no competing interests. We thank the staff and patients at the Mandalay General Hospital who participated in this study. We thank the Myanmar Ministry of Health and Sports for supporting this project. Last, we thank the Australian Department of Foreign Affairs and Trade for funding this project. All patients provided consent for this study. In patients who were too unwell, consent was obtained from close relatives. Download .pdf (.76 MB) Help with pdf files Supplementary File (PDF)

The Myanmar Snakebite Project is an Australian government (Department of Foreign Affairs and Trade) supported foreign aid project in collaboration with the Myanmar government with the aim of improving outcomes for snakebite patients in Myanmar. As part of the project a case record database was established to document prospective cases of snakebite presenting to Mandalay General Hospital, in Upper Myanmar. The study period was 12 months (1-2-2016 to 31-1-2017). Snake identity was based on a mixture of identified dead snakes brought with patients, doctor's clinical opinion and patient identification. 965 patients were enrolled during the 12 month period, of whom 948 were included for analysis. The male: female ratio was 1.58:1. Most cases involved bites to the lower limbs (82.5%) and adults involved in farm work, confirming snakebite as an occupational disease in this community. Motorised transport was by far the most common form of transport to health care and most patients sought care from the health system (87.7%), not traditional healers (11.5%) as their first point of contact. The officially promoted application of a pressure pad, bandage and immobilisation as first aid for snakebite was almost never used, while most patients used some form of tourniquet (92.0%). 85.4% of cases where a snake ID was listed were bitten by Russell's vipers. Russell's viper bites were responsible for all fatalities (9.8% of cases) and all cases of Acute Kidney Injury (AKI). For all cases, clinical features included local swelling (76.5%), local pain (62.6%), AKI (59.8%), incoagulable blood (57.9%), regional lymphadenopathy (39.8%), nausea/vomiting (40.4%), thrombocytopenia (53.6%), abdominal pain (28.8%), shock (11.8%), secondary infection (8.6%), panhypopituitarism (2.1%). AKI required renal replacement therapy (RRT) in 23.9% of cases, all ascribed to Russell's viper bite. Green pit viper bites were the next most common cause of bites (7.6%) and were associated with incoagulable blood (29%) and occasionally shock (5%) and local necrosis (3%), and in one case AKI not requiring RRT. In contrast to Russell's viper bites, green pit viper bite was most likely to occur in the home (49%). Some green pit viper patients were treated with Russell's viper antivenom (15%), presumably because they had incoagulable blood, although this antivenom is not effective against green pit viper envenoming. For the entire patient group, antivenom was given in 80.5% of cases. The most common indications were presence of coagulopathy/non-clotting blood (59.8%), local swelling (47.4%), oliguria/anuria (19.8%), heavy proteinuria (19.4%). A febrile reaction to antivenom was reported in 47.9% of cases, while anaphylaxis, occurred in 7.9% of cases.

Aquatic habitats within and adjacent to the boundary of flowing water possess unique hydraulic characteristics that vary from slow-flowing to rapidly circulating and turbulent eddies. In these natural streams the dynamics of hydraulic characteristics have been studied and described by understanding empirical relationships from open channel hydraulics. At the reach scale, mesohabitats described as pools, riffles, and glides consist of nonuniform-flow patterns that can be predicted, and in some cases recreated, by understanding hydraulic and channel relationships. At the microhabitat scale, individual streamflow lines and states of flow can be delineated and partially analyzed with nonuniform-flow equations. Aquatic organisms have adapted to these varying hydraulic conditions (see also Chapters 15 and 16Chapter 15Chapter 16). This chapter provides an introduction to the dynamics and hydraulics of flowing water. It provides the student with the basics for understanding the complex nature of flowing water that help form aquatic habitats and features directly affecting aquatic organisms.

Background: The mulga snake (Pseudechis australis) is the largest terrestrial venomous snake in Australia. It is capable of inflicting severe and occasionally fatal envenoming, but there have been few studies of P. australis bites.Objectives: To highlight and reinforce the main features of P. australis envenoming and to provide a clearer picture of the epidemiology of bites from this species.Methods: Selected case records kept by the Toxinology Dept. (Women's and Children's Hospital, Adelaide, Australia) were reviewed retrospectively to determine definite P. australis bites. Inclusion criteria: definite cases where the snake was identified by a competent person and/or lab specimens (bite site/urine) tested positive for "black snake" using CSL snake venom detection kit in a locality within the known range of P. australis, but without sympatry with other Pseudechis spp. Exclusion criteria: where the snake could not be clearly identified under criteria above. Epidemiological and clinical information was recorded and analysed for the definite cases.Results: A total of 27 cases were identified as definite P. australis bites; there were no fatalities. The median age was 35.5 years (IQR 51-23) and 80% of bites occurred in males. More bites occurred in the warmer months (Dec March) and in those handling/interfering with snakes. Seven people were bitten whilst asleep at night. 21/27 patients developed systemic envenoming (based on signs, symptoms and laboratory results) and 17 cases received antivenom. Local bite site pain (18) and swelling (17) were common as were nonspecific generalised symptoms such as nausea, vomiting and headache. Myotoxicity (11) and anticoagulant coagulopathy (10) occurred frequently; haemolysis was seen in fewer cases (3). Two patients developed local tissue injury around the bite site requiring further treatment.Conclusions: This study confirms previous reports about P. australis bites with respect to high rates of envenoming, commonly associated with pain and swelling and systemic effects of rhabdomyolysis and anticoagulant coagulopathy. Systemic envenoming, even severe cases, responds well to antivenom therapy. Compared to other Australian snakes, a high proportion of bites occur in people asleep at night. Medically significant local tissue injury around the bite site may occur and may be associated with inappropriate first-aid, particularly the vascular occlusive type. (C) 2014 Elsevier Ltd. All rights reserved.

BACKGROUND:The mulga snake (Pseudechis australis) is the largest terrestrial venomous snake in Australia. It is capable of inflicting severe and occasionally fatal envenoming, but there have been few studies of P. australis bites. OBJECTIVES:To highlight and reinforce the main features of P. australis envenoming and to provide a clearer picture of the epidemiology of bites from this species. METHODS:Selected case records kept by the Toxinology Dept. (Women's and Children's Hospital, Adelaide, Australia) were reviewed retrospectively to determine definite P. australis bites. INCLUSION CRITERIA:definite cases where the snake was identified by a competent person and/or lab specimens (bite site/urine) tested positive for "black snake" using CSL snake venom detection kit in a locality within the known range of P. australis, but without sympatry with other Pseudechis spp. EXCLUSION CRITERIA:where the snake could not be clearly identified under criteria above. Epidemiological and clinical information was recorded and analysed for the definite cases. RESULTS:A total of 27 cases were identified as definite P. australis bites; there were no fatalities. The median age was 35.5 years (IQR 51-23) and 80% of bites occurred in males. More bites occurred in the warmer months (Dec-March) and in those handling/interfering with snakes. Seven people were bitten whilst asleep at night. 21/27 patients developed systemic envenoming (based on signs, symptoms and laboratory results) and 17 cases received antivenom. Local bite site pain (18) and swelling (17) were common as were non-specific generalised symptoms such as nausea, vomiting and headache. Myotoxicity (11) and anticoagulant coagulopathy (10) occurred frequently; haemolysis was seen in fewer cases (3). Two patients developed local tissue injury around the bite site requiring further treatment. CONCLUSIONS:This study confirms previous reports about P. australis bites with respect to high rates of envenoming, commonly associated with pain and swelling and systemic effects of rhabdomyolysis and anticoagulant coagulopathy. Systemic envenoming, even severe cases, responds well to antivenom therapy. Compared to other Australian snakes, a high proportion of bites occur in people asleep at night. Medically significant local tissue injury around the bite site may occur and may be associated with inappropriate first-aid, particularly the vascular occlusive type.

•We provide a broad picture of the clinical features of Pseudechis australis envenoming.•We confirm high rates of envenoming, with pain, swelling and systemic effects.•A high proportion of bites occur in people asleep at night.•Medically significant local tissue injury may occur around the bite site.

Riffles and rapids may be added to channels for a variety of purposes, including increasing hydraulic complexity, stabilizing mobile bed streams, increasing aquatic habitat, or restoring fish passage. To increase hydraulic complexity, there are several options for introducing locally varied hydraulic conditions through the creation of riffles, rapids, runs, and pools. This involves increasing the frequency of transitions between several conditions of uniform, gradually varied and rapidly varied flow. To improve fish passage, riffles and rapids are normally designed as fish-passable hydraulic structures, often replacing traditional fixed drop structures or low dams in channelized streams. The provision of more diverse hydraulics and fish access may be a project objective, but the intricacies of specific aquatic habitat types are beyond commonly used one-dimensional open channel hydraulic equations. Consequently, reliance is placed on mimicking the hydraulics of preferred habitats surveyed in natural reference streams. The hydraulics observed in several preferred aquatic habitat types are broadly summarized, and a design method for riffles, runs, and pools with six project examples is presented.

AbstractGrowth hormone concentrations in juvenile cocho salmon were determined after intraperitoneal injections (yearlings; age 1+) and oral treatments (yearlings and fry; age 0+) with clonidine. Weights and lengths were also measured in coho juveniles fed diets supplemented with clonidine.Plasma growth hormone concentration increased significantly (78.2%) 24 h after injection. Yearlings fed clonidine showed a significant increase (136%) in plasma growth hormone after 14 days, and levels remained elevated through 28 days (116%) before dropping to control levels at 42 days. Coho yearlings fed clonidine showed no significant increase in growth compared to controls after 56 days of treatment, but coho fry were significantly heavier (33%) and longer (8%) after 70 days.

Circulating levels of plasma growth hormone were measured in juvenile coho salmon (Oncorhynchus kisutch) over a 24-h period. Growth hormone followed a circadian rhythm, peaking three times during 24 h. Peaks occurred at 10:00, 16:00, and 24:00, with the largest peak (24:00) representing a 575% increase in plasma growth hormone over a 2-h period (22:00–24:00).

The development and validation of a bioanalytical assay is described for the simultaneous analysis in human serum of tamoxifen, four of its main metabolites and three flavonoids, which are known constituents in alternative medicine and dietary supplements often used by breast cancer patients. The method has been fully validated at linear ranges covering steady-state serum concentrations in patients who receive therapeutic dosages of tamoxifen. The wide range also allows for quantification of large inter-patient fluctuations of flavonoid concentrations. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of a protein precipitation with acetonitrile using only 50 μL serum. The described method is simple, robust and reproducible with inter- and intra-assay accuracies within 85–115%. The applicability of the assay was demonstrated and it is now successfully used to study the in vivo pharmacokinetics of tamoxifen, its main metabolites and flavonoids in human serum of patients receiving tamoxifen.

The metabolism of cyclophosphamide was studied in vitro using isolated rat hepatocytes and mass spectrometry. The major product of primary oxidative metabolism in hepatocytes from phenobarbital treated rats was 4-hydroxycyclophosphamide, isolated as the O-ethyl derivatives, but dechloroethylation was also a substantial pathway. 4-Hydroxycyclophosphamide was converted mainly into carboxy phosphamide and the formation of 4-ketocyclophosphamide was a minor pathway. Evidence is presented that under certain conditions a substantial amount of an O-glucuronide of 4-hydroxycyclophosphamide was formed. The pattern of metabolism in hepatocytes otherwise resembled qualitatively that observed previously in vitro using subcellular fractions and in vivo, but quantitative differences were found. The metabolism of cyclophosphamide by hepatocytes resembles more closely that in vivo than does the metabolism in subcellular fractions, and hepatocytes should be the preferred in vitro system for studying the metabolism of anti-tumour agents.

Computer simulation of the full time course of an enzymatic reaction may be used to help define certain characteristics of the mechanism of reaction. In particular, such studies may be the best way to approach the kinetic properties of those enzymes which show hysteretic behavior. Furthermore, full time course studies may be used to differentiate between various mechanisms involving two substrates. For example, it is shown that different time courses may be obtained for a mechanism assuming ordered addition of substrates relative to one assuming a random addition of substrates. It is pointed out that the use of full time course studies may be applicable to a large number of mechanisms as a routine kinetic method of great utility for approaching the definition of such mechanisms. After a mechanism for an enzymatic reaction has been assumed, the full time course computer simulation program discussed here can also be used as a data-fitting program to obtain kinetic parameters for both the forward and reverse reactions.

The full time course of reduced coenzyme oxidation by glutamate dehydrogenase has been measured and the data have been compared with computer-simulated progress curves. It is shown that the oxidation of TPNH, at saturating concentrations of α-ketoglutarate and ammonium ion, follows a relatively simple mechanism but must include the formation of an abortive enzyme-TPN-substrate complex. The progress curve for the oxidation of DPNH is multiphasic and reflects the fact that DPNH may bind to a second site, inducing an isomerization of the enzyme which results in inhibition of the reaction. As a consequence of the fact that this isomerization is relatively slow, the kinetic properties using DPNH as coenzyme are dependent upon enzyme concentration. The rate constants for the induced isomerization and the dissociation constants for the abortive complex formation, for the reduced coenzyme binding to the active site, and for DPNH binding to the second site have been determined from fits of computer-calculated simulation curves to the experimental data.