Delivering solid treatments on shaky ground: Feasibility study of an online therapy for child anxiety in the aftermath of a natural disaster.

OBJECTIVE: To assess feasibility of online cognitive behaviour therapy (CBT) for children and adolescents with anxiety in the aftermath of a natural disaster. METHOD: 42 children and adolescents with clinical anxiety referred from primary care were invited to complete an internet CBT program (BRAVE-ONLINE). Outcome measures and assessment timelines were chosen to allow a comparison of the results with the program developers' randomised controlled trials. RESULTS: At 6-month post intervention, more than half (55%) of the 33 participants assessed, no longer met criteria for their primary anxiety disorder. The mean number of anxiety diagnoses dropped from 2.76 (SD = 0.85) at baseline to 1.06 (SD = 1.25) at follow-up (z = -4.51, p < .001). Participants' anxiety and mood symptoms reduced and health-related quality of life improved significantly by follow-up. Satisfaction ratings were moderate to high. On average, by 6-month follow-up, children and adolescents had completed 6 of 10 sessions and parents had completed 5/6 (child parent program) and 3/5 sessions (adolescent parent program). CONCLUSIONS: Following a natural disaster (the Canterbury earthquakes), children and adolescents showed clinically significant improvement in anxiety and mood when they used BRAVE-ONLINE. This approach was both feasible and acceptable to families and offered a solution when mental health services were under pressure.

US Preventative Services Task Force FRAX threshold has a low sensitivity to detect osteoporosis in women ages 50-64 years.

UNLABELLED: The US Preventative Services Task Force (USPSTF) recommends consideration for screening for osteoporosis in women under age 65 who have an estimated 10-year major osteoporotic fracture risk of 9.3 % or higher. We found that this threshold for osteoporosis screening in women ages 50-64 years old has a low sensitivity to detect osteoporosis. INTRODUCTION: The US Preventative Services Task Force (USPSTF) recommends consideration of dual-energy X-ray absorptiometry (DXA) in women under ages 50-64 with a major osteoporotic fracture (MOF) risk of 9.3 % or higher, as estimated by the fracture risk assessment tool (FRAX) tool. We assessed the performance of the 9.3 % MOF risk threshold for detecting osteoporosis and evaluated whether DXA indication appeared appropriate, based on USPSTF criteria and other risk factors, at our institution. METHODS: We performed a retrospective record review of women ages 50-64.5 years old to determine clinical factors and FRAX scores of women undergoing a DXA at our institution over a 6-month period after the USPSTF recommendations were released and evaluated the sensitivity and specificity of the 9.3 % MOF threshold to detect densitometric osteoporosis. Additionally, using the USPSTF criteria and several additional risk factors, we evaluated the extent of potentially inappropriate DXA use in women ages 50 to 64 years in a large primary care practice in an academic medical center. RESULTS: The analysis included 465 DXA tests. The overall sensitivity and specificity of a FRAX-calculated MOF risk ≥9.3 % was 37 and 74 %, respectively, for the detection of osteoporosis. The receiver operator characteristic curve (ROC) demonstrated an area under the curve of 0.58. Lowering the FRAX risk threshold to 5.5 % would increase the sensitivity of detecting osteoporosis in our population from 37 to 80 % while reducing the specificity from 74 to 27 %. Out of 465 DXAs, 371 (79.8 %) were classified as appropriately ordered per our pre-specified criteria. Of the 120 women with osteoporosis at the hip and/or spine based on T-score values of -2.5 or less, 14 DXAs (11.7 %) were classified as potentially inappropriate based on a FRAX-predicted MOF risk less than 9.3 % and lack of additional pre-specified risk factors. CONCLUSION: We found that the USPSTF-recommended MOF risk threshold of 9.3 % for osteoporosis screening in women ages 50-64 years old has a low sensitivity to detect osteoporosis.

Precision computerised cognitive behavioural therapy (cCBT) for adolescents with depression: a pilot and feasibility randomised controlled trial protocol for SPARX-UK.

BACKGROUND: A serious game called SPARX (Smart, Positive, Active, Realistic, X-factor thoughts), originally developed in New Zealand and incorporating cognitive behavioural therapy (CBT) principles, has been shown to help reduce symptoms of depression and anxiety in adolescents with mild to moderate depression in studies undertaken in Australasia. However, SPARX has never been trialled in the United Kingdom (UK), and there have been issues relating to low engagement when it has been used in a real-world context. AIMS: To conduct the first pilot and feasibility randomised controlled trial (RCT) in England to explore the use of SPARX in different settings. The trial will explore whether SPARX supported by an e-coach (assistant psychologists) improves adherence and engagement compared with self-directed (i.e. self-help) use. The trial results will be used to inform the optimal mode of delivery (SPARX supported vs. SPARX self-directed), to calculate an appropriate sample size for a full RCT, and to decide which setting is most suitable. METHODS: Following consultation with young people to ensure study suitability/appropriateness, a total of 120 adolescents (11-19 years) will be recruited for this three-arm study. Adolescents recruited for the study across England will be randomised to receive either SPARX with human support (from an e-coach), self-directed SPARX, or a waitlist control group. Assessments will be conducted online at baseline, week 4, and 8-10-week post-randomisation. The assessments will include measures which capture demographic, depression (Patient Health Questionnaire modified for adolescents [PHQ-A]) and anxiety (Revised Child Anxiety and Depression Scale [RCADS]) symptomatology, and health-related quality-of-life data (EQ-5D-Y and proxy version). Analyses will be primarily descriptive. Qualitative interviews will be undertaken with a proportion of the participants and clinical staff as part of a process evaluation, and the qualitative data gathered will be thematically analysed. Finally, feasibility data will be collected on recruitment details, overall study uptake and engagement with SPARX, participant retention, and youth-reported acceptability of the intervention. DISCUSSION: The findings will inform the design of a future definitive RCT of SPARX in the UK. If the subsequent definitive RCT demonstrates that SPARX is effective, then an online serious game utilising CBT principles ultimately has the potential to improve the provision of care within the UK's health services if delivered en masse. TRIAL REGISTRATION: ISRCTN: ISRCTN15124804. Registered on 16 January 2023, https://www.isrctn.com/ISRCTN15124804 .

Selective serotonin reuptake inhibitors (SSRIs) for depressive disorders in children and adolescents.

BACKGROUND: Depressive disorders are common in young people and are associated with significant negative impacts. Selective serotonin reuptake inhibitors (SSRIs) are often used, however, evidence of their effectiveness in children and adolescents is not clear. Furthermore, there have been warnings against their use in this population due to concerns about increased risk of suicidal ideation and behaviour. OBJECTIVES: To determine the efficacy and adverse outcomes, including definitive suicidal behaviour and suicidal ideation, of SSRIs compared to placebo in the treatment of depressive disorders in children and adolescents. SEARCH STRATEGY: We searched the CCDAN Trials Register, MEDLINE, PSYCHINFO and CENTRAL. Reference lists were checked, letters were sent to key researchers and internet databases searched. SELECTION CRITERIA: We included published and unpublished randomised controlled trials. DATA COLLECTION AND ANALYSIS: Two or three review authors selected the trials, assessed the quality and extracted trial and outcome data. We used a fixed-effect meta-analysis. The relative risk was used to summarise dichotomous outcomes and the mean difference to summarise continuous measures. MAIN RESULTS: Twelve trials were eligible for inclusion, with ten providing usable data. At 8-12 weeks, there was evidence that children and adolescents 'responded' to treatment with SSRIs (RR 1.28, 95% CI 1.17 to 1.41). There was also evidence of an increased risk of suicidal ideation and behaviour for those prescribed SSRIs (RR 1.80, 95% CI 1.19 to 2.72). Fluoxetine was the only SSRI where there was consistent evidence from three trials that it was effective in reducing depression symptoms in both children and adolescents (CDRS-R treatment effect -5.63, 95% CI -7.38 to -3.88), and 'response' to treatment (RR 1.86, 95% CI 1.49 to 2.32). Where rates of adverse events were reported, this was higher for those prescribed SSRIs. AUTHORS' CONCLUSIONS: Caution is required to interpret the results. First, there were methodological issues, including high attrition, issues regarding measurement instruments and clinical usefulness of outcomes, often variously defined across trials. Second, patients seen in clinical practice are likely to be more unwell, and at greater risk of suicide, compared to those in the trials, and it is unclear how this group would respond to SSRIs. This needs to be considered, along with the evidence of an increased risk of suicide related outcomes in those treated with SSRIs. It is unclear what the effect of SSRIs is on suicide completion. While untreated depression is associated with the risk of completed suicide and impacts on functioning, it is unclear whether SSRIs would modify this risk in a clinically meaningful way.

Circumvention of pleiotropic drug resistance in subcutaneous tumours in vivo with verapamil and clomipramine.

The development of pleiotropic drug resistance (PDR) in vivo in solid tumour models suggests that a similar process may occur in the clinic. A subline of the Ridgway osteogenic sarcoma (ROS)--a murine subcutaneously-growing solid tumour--with moderate resistance (1.5 fold) to actinomycin D was selected by repeated suboptimal treatment with this drug in vivo. This subline (ROS/ADX/G2) showed cross-resistance to vincristine (3.5 fold) and etoposide (over 5.1 fold) but not to doxorubicin. The resistance could in all cases be partly or completely overcome by treatment with non-cytotoxic doses of verapamil or clomipramine. Resistance to actinomycin in this model was associated with lower (up to 3.2 fold) drug accumulation into tumours which could be increased (up to 2.8 fold) by treatment with 25 micrograms/g verapamil. These data support clinical trials of the use of membrane-active agents to overcome PDR.

Evidence of impaired myocardial perfusion and abnormal left ventricular function during exercise in patients with isolated systolic narrowing of the left anterior descending coronary artery.

Seven men ranging in age from 35 to 63 years with a chest pain syndrome and cineangiographically documented systolic narrowing of the left anterior descending coronary artery underwent thallium-201 myocardial scintigraphy and gated cardiac blood pool imaging. Grade II (50 to 75 percent) systolic coronary arterial constriction was present in three patients and grade III constriction (greater than 75 percent) in four. Three of the four patients with grade III constriction had an exercise-induced perfusion abnormality in the thallium-201 scintigram and impaired left ventricular ejection fraction response during exercise. (In two patients the left ventricular ejection fraction did not change and in one patient it decreased.) Each of the three patients with grade II constriction had normal thallium-201 perfusion and a normal increase in ejection fraction during exercise. These data provide evidence of abnormal myocardial perfusion and impaired left ventricular function during exercise in patients with high grade systolic coronary arterial narrowing.

Psychiatric status of sexually abused children 12 months after disclosure of abuse.

OBJECTIVE: The assessment of psychiatric status of sexually abused children 12 months after the disclosure of recent sexual abuse. METHOD: Ninety-five children, aged from 4 through 16 years, were recruited to the study from a variety of agencies other than psychiatric units. Sixty-six (69.5%) were assessed for psychiatric diagnosis on DSM-III-R using data from parents, teachers, and children 12 months after disclosure of abuse. Abuse was extra- and intrafamilial. RESULTS: Overall 63.5% of the children warranted a diagnosis on Axis I. There was a wide range of diagnoses, with particularly high rates of oppositional defiant disorder (19.6%), post-traumatic stress disorder (18.2%), anxiety disorders (30.3%), depressive disorders (12.1%), and attention-deficit hyperactivity disorder (13.6%). Boys had a higher rate of diagnosis than girls. Abuse and social variables did not predict diagnoses but mothers' mental status rated on the General Health Questionnaire did. Subjects not located at follow-up were more often male and more likely to be socioeconomically disadvantaged. Thus the estimates of psychopathology here are likely to be conservative. CONCLUSIONS: This study highlights the need of sexually abused children for skilled long-term therapy tailored to individual presentation.

Methylphenidate and thioridazine in the treatment of intellectually subaverage children: effects on cognitive-motor performance.

Twenty-seven children (or smaller subgroups depending upon task difficulty and subject ability) with subaverage IQs took part in a double-blind, placebo-controlled, cross-over study of methylphenidate (0.4 mg/kg/day) and thioridazine (1.75 mg/kg/day). The children were tested for IQ performance, breadth of attention, and performance on a series of electronically controlled cognitive-motor tests. Methylphenidate improved accuracy on a memory task, reduced omission errors on an attentional task, and reduced seat movements on two tasks. Thioridazine failed to have any deleterious effects on IQ performance when subjects received reinforcers for correct answers. Thioridazine at the given dose did not adversely affect performance on any of the cognitive-motor performance tests. Methylphenidate appears likely to enhance sustained attention and motivation in appropriately selected children with mild developmental delays, whereas thioridazine at this modest dose does not appear to impair performance on most psychomotor tests.

Clinical effects of methylphenidate and thioridazine in intellectually subaverage children.

Thirty children with subaverage IQs and psychiatric diagnoses of attention deficit disorder and/or or conduct disorder took part in a double-blind study of placebo, methylphenidate, and thioridazine, which were given for 3 weeks each. The results showed a consistent and highly significant effect of methylphenidate in reducing teacher ratings of problem behavior. Parent ratings showed no behavioral effects for the group as a whole. An attentional model of stimulant drug response was used to divide subjects according to a cognitive maturity domain presumed to reflect selective attention. When divided according to breadth of attention, mental age, and IQ level, higher functioning subjects were found to show a generally favorable response to methylphenidate on both teacher and parent rating scales, whereas children of low functional level typically showed an adverse or indifferent response. The present data suggest that mental age and IQ may be important determinants of drug response; below a given level, there was a greatly reduced likelihood of responding positively. Clinical response to thioridazine was substantially less than the response to methylphenidate, with significant improvements confined to conduct and hyperactivity problems on teacher ratings.

Tumour cell resistance to anthracyclines--a review.

Resistance to anthracyclines is the major factor limiting their clinical utility. Laboratory studies using cultured experimental and human tumour cells have indicated that reduced intracellular drug accumulation is one important factor underlying resistance. In some systems this results from enhanced active drug efflux, a process which may be circumvented experimentally, for example by calcium antagonists. A specific glycoprotein which is produced in excess and is inherited has been identified in the cell membrane of certain anthracycline-resistant cells, while gene amplification with the appearance of double-minute chromosomes has been noted in others. Thus it is possible that anthracycline resistance arises following inherited changes in the cell membrane resulting in failure of drug accumulation. However, other possibilities exist, including differences in drug binding, either to the cell membrane or to nuclei, differences in metabolism to the semiquinone free radical, and differences in drug penetration related to tumour morphology. For each human tumour type the factor(s) involved may differ, but sufficient clues now exist to suggest that clinical testing of some of the therapeutic possibilities for circumventing anthracycline resistance may soon be appropriate.

Identification and characterisation in vitro of cells with a non-SCLC cell-like phenotype derived from a continuous SCLC cell line.

Two adherent sublines, H69V and H69VZ, have been isolated from the classic SCLC cell line NCI-H69. Significant morphological differences were observed between the parental and the derivative cell lines. While NCI-H69 grew as densely packed free floating cellular aggregates the derivative lines grew as a monolayer of epithelioid cells. The growth rates of both the derivative lines were faster than the parental line with doubling times closer to non-SCLC cell lines in the derivative lines. Both H69V and H69VZ either express very low levels or do not express neuroendocrine cell markers including L-dopa-decarboxylase (DDC), creatine kinase-BB isoenzyme (CK-BB), bombesin-like immunoreactivity (BLI), neuron specific enolase (NSE), and neurosecretory type dense core granules (DGCs), compared to the parental cell line. All the lines stained positive for epithelial markers such as CAM5.2. LDH isoenzyme and chromosome analyses confirmed the human origin of all the cell lines. Therefore, it appears that cell line NCI-H69 contains stem cell subpopulation capable of generating cells of both small and non-small cell like phenotypes.

Psychological and/or educational interventions for the prevention of depression in children and adolescents.

BACKGROUND: Depression is the fourth most important disease in the estimation of the burden of disease Murray 1996 and is a common problem with prevalence rates estimated to be as high as 8% in young people. Depression in young people is associated with poor academic performance, social dysfunction, substance abuse, suicide attempts, and completed suicide (NHMRC 1997). This has precipitated the development of programmes aimed at preventing the onset of depression. This review evaluates evidence for the effectiveness of these prevention programmes. OBJECTIVES: To determine whether psychological and/or educational interventions (both universal and targeted) are effective in reducing risk of depressive disorder by reducing depressive symptoms immediately after intervention or by preventing the onset of depressive disorder in children and adolescents over the next one to three years. SEARCH STRATEGY: The Cochrane Depression, Anxiety and Neurosis Group trials register (August 2002), MEDLINE (1966 to December Week 3 2002), EMBASE (1980 to January Week 2 2003), PsychInfo (1886 to January Week 2 2003) and ERIC (1985 to December 2002) were searched. In addition, conference abstracts, the reference lists of included studies, and other reviews were searched and experts in the field were contacted. SELECTION CRITERIA: Each identified study was assessed for possible inclusion by two independent reviewers based on the methods sections. The determinants for inclusion were that the trial include a psychological and/or educational prevention programme for young people aged 5 to 19 years-old, who did not meet DSM or ICD criteria for depression and/or did not fall into the clinical range on standardised, validated, and reliable rating scales of depression. DATA COLLECTION AND ANALYSIS: The methodological quality of the included trials was assessed by two independent reviewers according to a list of pre-determined criteria, which were based on quality ratings devised by Moncrieff and colleagues (Moncrieff 2001). Outcome data was extracted and entered into Revman 4.2. Means and standard deviations for continuous outcomes and number of events for dichotomous outcomes were extracted where available. For trials where the required data were not reported or could not be calculated, further details were requested from first authors. If no further details were provided, the trial was included in the review and described, but not included in the meta-analysis. Results were presented for each type of intervention: targeted or universal interventions; and educational or psychological interventions and if data were provided, by gender. Where possible data were combined in meta-analyses to give a treatment effect across all trials. Sensitivity analysis were conducted on studies rated as "adequate" or "high" quality, that is with a score over 22, based on the scale by Moncrieff et al (Moncrieff 2001). The presence of publication bias was assessed using funnel plots. MAIN RESULTS: Studies were divided into those that compared intervention with an active comparison or placebo (i.e. a control condition that resembles the intervention being investigated but which lacks the elements thought to be active in preventing depression) and those that used a "wait-list" or no intervention comparison group. Only two studies fell into the former category and neither showed effectiveness although one study was inadequately powered to show a difference and in the other the "placebo" contained active therapeutic elements, reducing the ability to demonstrate a difference from intervention. Psychological interventions were effective compared with non-intervention immediately after the programmes were delivered with a significant reduction in scores on depression rating scales for targeted (standardised mean difference (SMD) of -0.26 and a 95% confidence interval (CI) of -0.40 to -0.13 ) but not universal interventions (SMD -0.21, 95% CI -0.48, 0.06), with a significant effect maintained on pooling data (SMD -0.26, 95% CI -0.36, -0.15). While small effect sizes were reported, these were associated with a significant reduction in depressive episodes. The overall risk difference after intervention translates to "numbers needed to treat" (NNT) of 10. The most effective study is the targeted programme by Clarke (Clarke 2001) where the initial effect size of -0.46 is associated with an initial risk difference of -0.22 and NNT 5. There was no evidence of effectiveness for educational interventions. Reports of effectiveness for boys and girls were contradictory. The quality of many studies was poor, and only two studies made allocation concealment explicit. Sensitivity analysis of only high quality studies did not alter the results significantly. The only analysis in which there was significant statistical heterogeneity was the sub-group analysis by gender where there was variability in the response to different programmes for both girls and boys. For the most part funnel plots indicate findings are robust for short term effects with no publication bias evident. There are too few studies to comment on whether there is publication bias for studies reporting long-term (12-36 month) follow-up. REVIEWER'S CONCLUSIONS: Although there is insufficient evidence to warrant the introduction of depression prevention programmes currently, results to date indicate that further study would be worthwhile. There is a need to compare interventions with a placebo or some sort of active comparison so that study participants do not know whether they are in the intervention group or not, to investigate the impact of booster sessions to see if effectiveness immediately after intervention can be prolonged, ideally for a year or longer, and to consider practical implementation of prevention programmes when choosing target populations. Until now most studies have focussed on psychological interventions. The potential effectiveness of educational interventions has not been fully investigated. Given the gender differences in prevalence, and the change in these that occurs in adolescence with a disproportionate increase in prevalence rates for girls, it is likely that girls and boys will respond differently to interventions. Although differences have been reported in studies in this review the findings are contradictory and a more definitive delineation of gender specific responses to interventions would be helpful.

Adolescents' perceptions of a health survey using multimedia computer-assisted self-administered interview.

OBJECTIVE: To ascertain young people's perceptions of an adolescent health survey when administered by multimedia computer assisted self-administered Interview (M-CASI) through analysis of (1) questionnaire item responses and (2) focus group interviews. SETTING: Auckland, New Zealand, 1999. STUDY TYPE: Pilot testing of a 488-item branching questionnaire delivered using a youth-oriented and user-friendly M-CASI interface in a variety of settings using both desktop and laptop computers. Post pilot focus groups of participants identifying their perceptions and experiences of the survey. SAMPLE: 110 school students aged 12 to 18 years. RESULTS: The mean number of questions answered by participants was 316 with the median time to completion being 48 minutes. On average 65% of the total number of questions were seen and of these 1.5% were deliberately not answered. A high level of acceptability and enjoyment of M-CASI was found in the analysis of focus group responses and agreed with the item responses relating to M-CASI within the questionnaire itself. Participants identified privacy and confidentiality as being particularly important for the honesty of their responses. The passive matrix screens of the computers were popular as they could only be viewed from in front. CONCLUSIONS: M-CASI is an acceptable instrument for the administration of a youth health survey. Laptop computers with passive matrix screens are able to enhance perceptions of privacy and confidentiality, which may improve honesty of responses. IMPLICATIONS: M-CASI is now feasible and offers advantages in health surveying.

Mechanism of actinomycin D-induced resistance in Ridgway osteogenic sarcoma: an ultrastructural study.

The ultrastructural appearances of actinomycin D treated sensitive and resistant sublines of Ridgway osteogenic sarcoma (ROS) have been correlated with the suggested mechanism of drug-induced resistance. The resistant subline (designated ROS/ADX/G2) was developed by repeated suboptimal treatment of tumor bearing animals and passage of the fastest growing tumors. In the present experiments, animals bearing sensitive and resistant tumors were given a single intraperitoneal injection of actinomycin D (0.3 microgram/g) and examined at 1, 6 and 24 h after injection. The principal effect of actinomycin D treatment in both cell lines was the development of nucleolar segregation. This change, however, followed a different time-scale in each case, appearing more prominently at an early stage and returning more quickly to normal in the actinomycin D resistant cell line. These findings can be interpreted as being in agreement with the suggestion that reduced drug retention or an increased rate of detoxification provides the mechanism of acquired resistance.

Early intervention for schizophrenic disorders. Implementing optimal treatment strategies in routine clinical services. OTP Collaborative Group.

BACKGROUND: Early detection and intervention in schizophrenic disorders is an important challenge for psychiatry. METHOD: Review of literature on effective biomedical and psychosocial intervention strategies. RESULTS: Comprehensive programmes of drug and psychosocial interventions with adults who show early signs and symptoms of schizophrenic disorders may contribute to a lower incidence and prevalence of major episodes of schizophrenia. These programmes combine early detection of psychotic features by primary care services, with close liaison with mental health professionals. Long-term monitoring of signs of recurrence, with further intervention, appears essential to maintain these benefits. CONCLUSIONS: Field trials demonstrate that effective early treatment strategies can be routinely applied in clinical practice.

A pilot study to examine the experiences and attitudes of women with breast cancer towards one versus two-step axillary surgery.

PURPOSE: To elicit the views, experiences and preferences of women with clinically node negative breast cancer towards intra-operative sentinel lymph node biopsy (SLNB) analysis. METHODS: Focus groups with 14 women with breast cancer from two UK centres; one group had undergone the standard practice of waiting two weeks for results of their axillary surgery, the other had experienced the intra-operative SLNB analysis. RESULTS: Women generally were unaware about their lymph nodes, what their function is and how they are removed. Preference was indicated for intra-operative sentinel lymph node biopsy (SLNB) analysis provided clear descriptions were given about the risk of experiencing false negative and false positive results. DISCUSSION: Adopting an intra-operative analysis technique of axillary nodes was viewed as an excellent option by women from both centres. The immediacy of knowing the results was seen as a great advantage for their physical and psychological well being and more cost effective.

Disposition kinetics of adriamycin, adriamycinol and their 7-deoxyaglycones in AKR mice bearing a sub-cutaneously growing ridgway osteogenic sarcoma (ROS).

Disposition kinetics of Adriamycin (ADR), adriamycinol (AOL) and their 7-deoxyaglycones (ADR-DONE and AOL-DONE) have been studied in AKR mice bearing a s.c. growing ROS tumour after i.v. administration of 10 mg/kg. ADR and its metabolites were extracted from tissues by two different methods, separated and identified by HPLC. Tissue 7-deoxyaglycones were isolated, purified and then identified by HPLC, TLC and mass spectrometry. Kinetic profiles of ADR showed rapid equilibration of the drug with well perfused tissues but a slower and complex equilibration of the drug with the ROS tumour. Serum and tissue profiles of AOL were similar to the parent drug. From the kinetic profiles of the 7-deoxyaglycones it appeared that in the tissues their formation was rapid, with ADR-DONE always appearing first. Maximum concentrations of ADR-DONE were reached in the liver and heart only 10 min after drug administration. Estimated half lives of ADR-DONE were in liver, 1.1 hr and in heart, 2.8 hr and for AOL-DONE in liver, 5.4 hr, in heart, 5.1 hr and in serum, 4.1 hr.

The mechanism of rabbit muscle phosphofructokinase at pH8.

The mechanism of rabbit muscle phosphofructokinase was investigated by measurement of fluxes, isotope trapping and steady-state velocities at pH8 in triethanolamine/HCl buffer with 4 mM free Mg2+. Most observations were made at I0.2. The ratio Flux of fructose 1,6-bisphosphate----fructose 6-phosphate/Flux of fructose 1,6-bisphosphate----ATP at zero ATP concentration increased hyperbolically from unity to about 3.2 as the concentration of fructose 6-phosphate was increased. Similarly, the ratio Flux of fructose 1,6-bisphosphate----ATP/Flux of fructose 1,6-bisphosphate----fructose 6-phosphate at zero fructose 6-phosphate concentration increased from unity to about 1.4 as the concentration of ATP was increased. The addition of substrates must therefore be random, whatever the other aspects of the reaction. Further, from the plateau values of the ratios, it follows that the substrates dissociate very infrequently from the ternary complex and that at a low substrate concentration 72% of the reaction follows the pathway in which ATP adds first to the enzyme. Isotope-trapping studies with [32P]ATP confirmed that ATP can bind first to the enzyme in rate-limiting step and that dissociation of ATP from the ternary complex is slow in relation to the forward reaction. No isotope trapping of [U-14C]-fructose 6-phosphate could be demonstrated. The ratios Flux of ATP----fructose 1,6-bisphosphate/Flux of ATP----ADP measured at zero ADP concentration and the reciprocal of the ratio measured at zero fructose 1,6-bisphosphate concentration did not differ significantly from unity. Calculated values for these ratios based on the kinetics of the reverse reaction and assuming ordered dissociations of products or a ping-pong mechanism gave values very significantly greater than unity. These findings exclude an ordered dissociation or a substantial contribution from a ping-pong mechanism, and it is concluded that the reaction is sequential and that dissociation of products is random. Rate constants were calculated for the steps in the enzyme reaction. The results indicate a considerable degree of co-operativity in the binding between the two substrates. The observations on phosphofructokinase are discussed in relation to methods of measurement and interpretation of flux ratios and in relation to the mechanism of other kinase enzymes.