Empirically derived formulae for calculation of age- and region-related levels of iron, copper and zinc in the adult C57BL/6 mouse brain.

Metal dyshomeostasis is associated with neurodegenerative disorders, cancers and vascular disease. We report the effects of age (range: 3 to 18 months) on regional copper, iron and zinc levels in the brain of the C57BL/6 mouse, a widely used inbred strain with a permissive background allowing maximal expression of mutations in models that recapitulate these disorders. We present formulae that can be used to determine regional brain metal concentrations in the C57BL/6 mouse at any age in the range of three to eighteen months of life. Copper levels in the C57BL/6 mouse adult brain were highest in the striatum and cerebellum and increased with age, excepting the cortex and hippocampus. Regional iron levels increased linearly with age in all brain regions, while regional zinc concentrations became more homogeneous with age. Knockdown of the copper transporter Ctr1 reduced brain copper, but not iron or zinc, concentrations in a regionally-dependent manner. These findings demonstrate biometals in the brain change with age in a regionally-dependent manner. These data and associated formulae have implications for improving design and interpretation of a wide variety of studies in the C57BL/6 mouse.

Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System.

Sirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.

Mucins and CD56 as markers of tumour invasion and prognosis in periampullary cancer.

BACKGROUND: This study investigated the association of mucins and cluster of differentiation (CD) 56 with vascular and perineural invasion and survival in patients with periampullary cancer. METHODS: Immunohistochemical staining was performed on formalin-fixed pancreatic tissue microarrays (cancer, chronic pancreatitis and normal pancreatic tissue) constructed from 126 pancreatic resections (cancer, 104; chronic pancreatitis, 22). Mucin (MUC) 1, MUC4 and MUC5AC expression was quantified using the immunohistochemical score (range 0-300), MUC3 expression was described as membranous or cytoplasmic, and expression of CD56, MUC2 and MUC6 as present or absent. RESULTS: In cancers, vascular invasion correlated with overexpression (immunohistochemical score of 100 or more) of MUC1 (P = 0.003) and presence of MUC6 (P = 0.024), and perineural invasion correlated with overexpression of MUC5AC (P = 0.015). Reduced survival was observed with overexpression of MUC4 (P = 0.032) and MUC5AC (P = 0.048), membranous expression of MUC3 (P = 0.048), and presence of CD56 (P = 0.041). Perineural invasion also correlated with CD56 expression (P = 0.001). Overexpression of MUC4 and MUC5AC correlated with tumour recurrence (P = 0.001 and P = 0.034 respectively). Multivariable analysis identified membranous expression of MUC3 (P = 0.023), lymphatic invasion (P = 0.015) and perineural invasion (P = 0.004) as independent predictors of poor survival. CONCLUSION: Mucins and CD56 may be markers of prognosis in patients with periampullary cancer.

The effect of jaundice on the generation of anti-gastrin antibodies in G17DT immunized patients with advanced pancreatic cancer.

AIM: The aim of this study was to determine the ability of G17DT to generate anti-gastrin antibodies in jaundiced patients with biliary obstruction due to advanced pancreatic cancer. METHODS: G17DT was administered to 41 patients with advanced pancreatic adenocarcinoma by intramuscular (i.m.) injection at a dose of 250mcg at weeks 0, 1 and 3 of the study. RESULTS: Thirty-five of 41 patients participating in the study were categorized as responders in terms of their gastrin-17 antibody response. There was no correlation between the maximum G17 antibody response and the bilirubin level at either week 0 or week 12. The median survival of patients from the time of the first injection of G17DT was 204 days with 25% of patients surviving for or=305 days. CONCLUSION: This study shows that G17DT administered to jaundiced patients with advanced pancreatic cancer is immunogenic and well tolerated.

Inhibition of matrix metalloproteinase activity and growth of gastric adenocarcinoma cells by an angiotensin converting enzyme inhibitor in in vitro and murine models.

INTRODUCTION: Angiotensin converting enzyme (ACE) shares structural homology with the matrix metalloproteinase family of proteolytic enzymes (MMPs) responsible for degradation of the extracellular matrix (ECM). ACE inhibitors have been reported to protect against cancer in patients. The aim of this study was to determine whether the ACE inhibitor, captopril, could impair the activity of MMPs and impact on tumour invasion and growth in a cell line and murine model. METHODS: For proof of principle, the protein activity of human MMP-2 and MMP-9 produced by the HT1080 fibrosarcoma cell line was detected using gelatin zymography. Gene expression was determined by real time reverse transcriptase PCR and tumour cell invasion using Matrigel invasion chambers. The effect of captopril on the in vivo growth of MGLVA-1 human gastric adenocarcinoma xenografts was evaluated in a nude mouse model. RESULTS: Captopril inhibited activity of secreted MMP-9 and MMP-2, however, gene expression in HT1080 remained unaltered. Invasion of HT1080 cells was inhibited by 48% (p<0.001). Tumour size was reduced by 40-50% with 0.4 mg/ml captopril (p<0.01) and when combined with cisplatin the inhibition increased to 71% (p<0.05). DISCUSSION: ACE inhibitors inhibit the activity of secreted MMP-2 and -9 by a mechanism similar to synthetic MMP inhibitors. ACE inhibitors have previously been shown to inhibit tumour growth, however; this is the first study to demonstrate inhibition of a human gastric xenograft, both alone and in combination with cisplatin. These results support further investigation into the anticancer effects of ACE inhibitors.

Relationships between structural and functional measures of nutritional status in a normally nourished population.

BACKGROUND AND AIMS: Both anthropometric and functional measurements have been used in nutritional assessment and monitoring. Hand dynamometry is a predictor of surgical outcome and peak expiratory flow rate has been used as an index of respiratory muscle function. This study aims to measure in normal subjects the relationship between anthropometric measurements, voluntary muscle strength by hand grip dynamometry and respiratory muscle function by peak expiratory flow rate. METHODS: Ninety-eight subjects (46 male, 52 female) with a mean age of 45.9 years were studied. Hand grip strength was measured in the dominant and non-dominant hands with a portable strain-gauge dynamometer. Peak expiratory flow rate was measured using a mini-Wright peak flow meter. Three readings were taken, each 1 min apart, and the average recorded. Midarm muscle circumference (MAMC) was derived from triceps skin fold thickness and midarm circumference (MAC) using standard anthropometric techniques. Statistical relationships were measured with Pearson's coefficient of correlation. RESULTS: In both sexes there was significant correlation between hand grip strength in the dominant and non-dominant hands and peak expiratory flow rate (P<0.001). In men, there was a positive correlation between MAMC, hand grip strength (P<0.001) and peak expiratory flow rate (P<0.001). In women muscle function correlated with height (P<0.001) but not MAMC (P>0.05). CONCLUSIONS: In normal subjects bedside tests of skeletal and respiratory muscle function correlated with each other in both sexes, and with muscle mass in men but not in women.

A practical posture for hand grip dynamometry in the clinical setting.

BACKGROUND & AIMS: The optimal testing position for hand grip strength, which is a useful functional measure of nutritional status, is open to debate. We therefore examined the systematic difference between different postures in order to establish a methodology that is clinically relevant, easy to perform and reproducible. METHODS: Grip strength was measured in the dominant and non-dominant hands with a strain gauge dynamometer in three positions: lying at 30 degrees in bed with elbows supported, seated in an armchair with elbows supported and in a chair with elbows unsupported. The average of three readings made in each position, each 1 min apart, was recorded. RESULTS: 55 normally nourished subjects (26 male) were studied. Mean (95% CI) grip strengths measured in the dominant hand with the subject in bed, sitting in an armchair and sitting in a chair were 45.7 (42.3-49.2), 46.3 (42.9-49.8) and 48.5 (45.4-51.7) kg, respectively for males. Corresponding values for females were 29.4 (27.0-31.8), 29.3 (26.8-31.9) and 31.6 (28.8-34.3) kg. There was no significant difference (Student t-paired test) between measurements made in bed and on an armchair (P = 0.49), but the measurements made in a chair were significantly higher than those made in bed (P = 0.001) and in an armchair (P = 0.004). No statistical difference was present, comparing the three separate measurements in each position (Student t-paired test). CONCLUSIONS: Measurement of grip strength using hand dynamometry is reproducible and consistent. As all patients are not able to sit in a chair with elbows unsupported, in clinical practice it is more practicable to perform hand dynamometry with the elbows supported in a bed or armchair.

The 'two-week' target for the diagnosis of pancreatic carcinoma: an achievable aim?

AIM: The aim of this study was to evaluate the current diagnostic process for patients with pancreatic cancer in a University teaching hospital and to determine whether the 'two-week' target for rapid assessment was being met. METHODS: The notes of all patients with pancreatic cancer from June 1998 to June 2000 were reviewed to determine the time to diagnosis, investigations and procedures performed, number of admissions, length of hospital stay and survival RESULTS: There were 146 patients in total with a median (range) age of 71 (38-92) years. 18 (12%) had resectable lesions, while the remaining 128 patients had 134 palliative interventions (33 surgical; 101 radiological or endoscopic). The median number of hospital admissions for each patient was 2 (range 1-6) with a median length of hospital stay of 9 days (range 1-35 days). The median (IQR) time to diagnosis was significantly less in the jaundiced patients [7 (6-10) days vs. 32 (18-46) days, P<0.0001]. There was no significant correlation between age and time to diagnosis (r=0.08, P=0.36). There were 105 (72%) deaths in the study population, 82 in the jaundiced group and 23 in the non-jaundiced group. There was no significant difference in median (IQR) duration from referral to death in the jaundiced and non-jaundiced groups [59.5 (18-175) days vs. 35 (16-137) days, P=0.45]. CONCLUSIONS: A diagnosis within 14 days was achieved in 77% of patients. Patients with jaundice were more likely to have an earlier diagnosis than those without jaundice but this had no impact on survival.

Host immune responses and intestinal permeability in patients with jaundice.

BACKGROUND: Systemic endotoxaemia is implicated in the development of complications associated with obstructive jaundice. The aims of these studies were to assess the systemic immune response to intervention in patients with jaundice and to compare the effects of surgical and non-surgical biliary drainage on host immune function and gut barrier function. METHODS: In the first study, 18 jaundiced and 12 control patients were studied to assess systemic immune responses before and after intervention. In the second study, immune responses and gut barrier function were assessed following surgical and non-operative biliary decompression in 45 patients with jaundice. RESULTS: Endotoxin antibody concentrations fell significantly in patients with jaundice immediately after surgical intervention, but not after non-operative biliary drainage. This decrease was associated with a significant increase in serum P(55) soluble tumour necrosis factor (sTNF) receptor concentration (5.3 versus 10.5 ng/ml; P < 0.001), urinary excretion of P(55) TNF receptors (21.4 versus 78.8 ng/ml; P = 0.002) and intestinal permeability (lactulose : mannitol ratio 0.032 versus 0.082; P = 0.048). Intestinal permeability was significantly increased in patients with jaundice compared with controls (0.033 versus 0.015; P = 0.002). CONCLUSION: These data suggest that obstructive jaundice is associated with impaired gut barrier function and activation of host immune function that is exacerbated by intervention. Surgery causes an exaggerated pathophysiological disturbance not seen with non-operative biliary drainage procedures.

Surgical sepsis: dysregulation of immune function and therapeutic implications.

Sepsis is defined clinically as the systemic inflammatory response of the host to the documented systemic infection. The pathophysiological disturbance involves both the innate and adaptive immune systems encompassing cellular immunity, humoral components and the complement system. Dendritic cells (antigen-presenting cells) are key cells involved in the regulation of the immune response in sepsis, in particular in activating T cells and especially inducing the production and secretion of specific cytokines. These are the main mediators in establishing prominent disturbances of inflammation in patients with sepsis. The clinical features of the sepsis syndrome may vary from minor clinical disturbances to severe multiple organ failure and death of the host. Appropriate therapeutic strategies for patients with sepsis utilise conventional therapy and new novel forms of treatment, which are showing promise for the future.

Peri-operative fluid and electrolyte management: a survey of consultant surgeons in the UK.

BACKGROUND: Current peri-operative fluid and electrolyte management in the UK may be suboptimal. We assessed the attitudes of consultant surgeons to fluid and electrolyte prescribing and gathered suggestions for improvement in education on the subject. METHODS: A postal questionnaire survey was sent to 1091 Fellows of the Association of Surgeons of Great Britain and Ireland. Of the 730 (67%) replies, 20 were invalid or incomplete, and 710 (65%) questionnaires were analysed. Outcome measures included provision of guidelines and teaching to junior staff on fluid and electrolyte prescribing, appropriateness of fluid management and suggestions to improve standards. RESULTS: Junior staff were given written guidelines in 22% of instances. Only 16% of respondents felt that their preregistration house officers (PRHOs) were adequately trained in the subject before joining the firm; 15% also stated that PRHOs did not receive much training on their firm. 65% felt that fluid balance charts were accurately maintained, nursing shortages being the commonest reason for inaccuracies. Only 30% felt that postoperative patients were receiving appropriate amounts of water, sodium and potassium. Respondents who had been consultants for > 5 years were more likely to prefer erring on the side of under-replacement of fluid than those who were consultants for 5 years (63% versus 47%, P < 0.0005). Suggestions for improvement in education included problem-oriented ward rounds, written guidelines, and discussion of patient scenarios. CONCLUSIONS: Consultant surgeons feel that present practice in peri-operative fluid management is unsatisfactory. Higher standards within clinical governance and risk management may be achieved by focused practical training combined with formal written guidelines.

Accentuated oxidative stress following reperfusion injury in diabetic rats.

BACKGROUND: This study investigates whether diabetes mellitus accentuates hind limb ischemia-reperfusion injury. METHODS: Male Wistar rats rendered diabetic (n=40) following injection of streptozotocin were compared to non-diabetic control rats (n=30). Each group was divided into sham, 4 hrs of hind limb ischemia, 4 hrs of ischemia followed by 10, 30 or 60 min of reperfusion. Plasma concentrations of an end-product of lipid peroxidation [malondialdehyde (MDA)] and antioxidants (vitamins A and E) were measured together with the resting membrane potential (RMP) of the gastrocnemius muscle. RESULTS: Following reperfusion, the diabetic group showed greater and more persistent elevation of MDA and greater reduction of antioxidants. This was associated with reduction in the RMP only in the diabetic group. There was significant correlation between MDA level and the RMP in both groups of animals. CONCLUSIONS: These results indicate that oxidative stress following reperfusion injury is greater in the presence of diabetes mellitus. This may lead to a decrease in the RMP and increase in the vascular permeability, which may be associated with more complications.

Reproducibility and normal ranges for gastric emptying in normal volunteers using a test meal designed for post-operative patients.

Scintigraphy is the current gold standard for the quantification of gastric emptying; however, results vary with meal composition. We modified a dual phase meal for administration to patients in the early post-operative period and aimed to test the reproducibility of the method, to obtain normal ranges and to compare these with previous data. Twenty healthy volunteers (10 male, 10 female), studied prospectively, were compared with 10 historical male volunteers. Each volunteer was studied twice (Test 1 at day 0 and Test 2 at day 7-10). After an overnight fast, subjects had Meal A consisting of a 60 g pancake labelled with 3 MBq of non-absorbable 99mTc-ion exchange resin and 100 ml of water labelled with 0.5 MBq of non-absorbable 111In-diethylenetriamine pentaacetic acid (111In-DTPA). Anterior and posterior gamma camera images of the stomach were obtained every 20 min for 3 h. The time for 50% emptying (T50) was derived from time-activity curves. Data obtained for males were compared with historical data using a similar technique with Meal B, consisting of two pancakes and a 200 ml milkshake labelled with identical amounts of radioisotopes. The mean (95% CI) T50 values for solid phase emptying for males and females using Meal A, and for historical males using Meal B, were 51.1 min (44.1-58.1), 58.6 min (52.7-64.5) and 128.9 min (112.8-145.1), respectively. Corresponding figures for the liquid phase were 33.2 min (26.1-40.3), 50.2 min (38.4-62.1) and 30.7 min (21.4-39.9). Bland-Altman plots for each phase showed good agreement between Tests 1 and 2 for Meal A. The modified test meal gave reproducible results in healthy volunteers; however, solid phase emptying was significantly faster than that of the bulkier test meal in historical subjects.

Audit of acute pancreatitis management: a tale of two hospitals.

BACKGROUND: A case note audit of existing practices in the management of acute pancreatitis (AP) at a district general hospital (DGH) and a teaching hospital was undertaken to determine if practices were in accordance with published UK guidelines. METHODS: Casenotes of all adults admitted with AP over a period of one year at the two hospitals were reviewed. RESULTS: Ninety-five patients were treated for AP at the teaching hospital and 52 at the DGH. The age, sex and aetiological distributions at the two hospitals were similar. Fifteen (15.8%) patients at the teaching hospital and eight (15.3%) at the DGH had severe AP. Four patients died at each hospital. Prognostic Glasgow criteria tests (excluding LDH) were completed within 48 hours in 43% patients at the teaching hospital and 48% at the DGH. Five of the twenty-five cholecystectomies at the teaching hospital and 4/18 at the DGH were performed within four weeks after admission with AP. CONCLUSION: Audit of current practice has highlighted deficiencies at many levels compared with current evidence-based guidelines, although this has not resulted in unexpected mortality. It remains to be seen whether new measures to aid compliance with guidelines will result in improvement in morbidity and mortality.

Body water compartment measurements: a comparison of bioelectrical impedance analysis with tritium and sodium bromide dilution techniques.

BACKGROUND AND AIMS: This study was undertaken to assess the comparability of body water compartment estimates in healthy volunteers using single and dual frequency bioelectrical impedance analysis (BIA) with established reference methods of tritium and NaBr dilution. METHODS: Total body water (TBW) was estimated in 10 healthy volunteers using single frequency (50 kHz) BIA (Bodystat 1500), dual frequency (5 and 200 kHz) BIA (Bodystat Dualscan 2005) and tritium dilution. Extracellular water (ECW) was measured with dual frequency BIA and NaBr dilution. BIA was performed using distal tetrapolar electrodes in the supine position. Venous blood was sampled for measurement of background concentrations of tritium and NaBr using a beta counter and high performance liquid chromatography respectively. 10 ml tritiated water (3.7 MBq) and 50 ml 5% NaBr solution were then injected intravenously and blood samples taken from the opposite arm every 45 min for 4.5 h for estimation of concentrations of tritium and NaBr. RESULTS: There was good correlation (r(2)=0.76) between estimates of ECW using dual frequency BIA and NaBr dilution, with the former overestimating ECW by approximately 1 L. However, this difference varied systematically with body weight. Although TBW measurements obtained by single and dual frequency BIA correlated well with estimates using tritium dilution (r(2)=0.96 and 0.95 respectively), single frequency BIA underestimated TBW by approximately 1 L and dual frequency BIA by approximately 5 L compared to tritium dilution. CONCLUSION: TBW measurements obtained using the single frequency BIA device were more accurate than those obtained using the dual frequency BIA device. Dual frequency BIA provided a reasonably accurate estimate of ECW.

Dilution and redistribution effects of rapid 2-litre infusions of 0.9% (w/v) saline and 5% (w/v) dextrose on haematological parameters and serum biochemistry in normal subjects: a double-blind crossover study.

Although hypoalbuminaemia after injury may result from increased vascular permeability, dilution secondary to crystalloid infusions may contribute significantly. In this double-blind crossover study, the effects of bolus infusions of crystalloids on serum albumin, haematocrit, serum and urinary biochemistry and bioelectrical impedance analysis were measured in healthy subjects. Ten male volunteers received 2-litre infusions of 0.9% (w/v) saline or 5% (w/v) dextrose over 1 h; infusions were carried out on separate occasions, in random order. Weight, haemoglobin, serum albumin, serum and urinary biochemistry and bioelectrical impedance were measured pre-infusion and hourly for 6 h. The serum albumin concentration fell in all subjects (20% after saline; 16% after dextrose) by more than could be explained by dilution alone. This fall lasted more than 6 h after saline infusion, but values had returned to baseline 1 h after the end of the dextrose infusion. Changes in haematocrit and haemoglobin were less pronounced (7.5% after saline; 6.5% after dextrose). Whereas all the water from dextrose was excreted by 2 h after completion of the infusion, only one-third of the sodium and water from the saline had been excreted by 6 h, explaining its persistent diluting effect. Impedances rose after dextrose and fell after saline (P<0.001). Subjects voided more urine (means 1663 and 563 ml respectively) of lower osmolality (means 129 and 630 mOsm/kg respectively) and sodium content (means 26 and 95 mmol respectively) after dextrose than after saline (P<0.001). While an excess water load is excreted rapidly, an excess sodium load is excreted very slowly, even in normal subjects, and causes persistent dilution of haematocrit and serum albumin. The greater than expected change in serum albumin concentration when compared with that of haemoglobin suggests that, while dilution is responsible for the latter, redistribution also has a role in the former. Changes in bioelectrical impedance may reflect the electrolyte content rather than the volume of the infusate, and may be unreliable for clinical purposes.

Problems with solutions: drowning in the brine of an inadequate knowledge base.

BACKGROUND & AIMS: We undertook a telephone questionnaire to determine current fluid prescribing practices and relevant knowledge among surgical preregistration house officers (PRHOs) and senior house officers (SHOs) working in 25 British hospitals. METHODS: One hundred PRHOs were surveyed within 10 days of starting their first job (Group A). Fifty other PRHOs were surveyed 6-8 weeks after starting their first job(Group B) along with 50 surgical SHOs (Group C). Outcome measures included responsibility for prescribing, knowledge of the composition of common intravenous fluids and the principles governing their use. RESULTS: PRHOs were responsible for prescribing in 89% of instances. Only 56% of respondents stated that fluid balance charts were checked on morning ward rounds. Less than half were aware of the sodium content of 0.9% saline or the daily sodium requirement. Although potassium supplements were usually correct, 25% of respondents prescribed two or more litres of 0.9% saline per day, which is far in excess of normal requirements. Although SHOs were more confident (P<0.0001), there was no significant difference between the three groups for most responses. CONCLUSIONS: Inadequate knowledge and suboptimal prescribing of fluid and electrolytes is common. Undergraduate and postgraduate training in this basic patient management skill needs improvement, with particular emphasis on the practical aspects.

Management of pancreatic pseudocysts.

BACKGROUND: This review analyses the outcome for patients with acute and chronic pancreatic pseudocysts managed in two major referral centres. PATIENTS AND METHODS: From 1987 to 1997, 33 patients were treated with either acute (n = 19) or chronic (n = 14) pseudocysts. Procedures performed included cystgastrostomy (64%), cystduodenostomy (6%), cystjejunostomy (3%), distal pancreatectomy with resection of pseudocyst (12%), laparotomy with external drainage (9%), endoscopic transpapillary stenting (3%) and endoscopic pancreatic duct sphincterotomy with percutaneous drainage of the pseudocyst (3%). RESULTS: All patients had resolution of their pseudocyst and no patient developed recurrence. There were no deaths in this series. There was a 9% incidence of major complications and a 21% incidence of minor complications. Outcome was excellent in 63% and good in 27% of patients. Two patients (6%) had persistent chronic pain and one patient (3%) had evidence of exocrine pancreatic insufficiency with malabsorption. CONCLUSIONS: Surgical internal drainage of pancreatic pseudocysts can be performed safely with low morbidity and mortality provided patients are carefully selected and their medical management is optimized. Although minimally invasive techniques now offer a variety of treatment options, open surgical drainage is still indicated for a significant number of cases.