Melatonin profiles during the third trimester of pregnancy and health status in the offspring among day and night workers: A case series.

Successful pregnancy requires adaptation in maternal physiology. During intrauterine life the mother's circadian timing system supports successful birth and postnatal development. Maternal melatonin is important to transmit circadian timing and day length to the fetus. This study aims to describe the third trimester of pregnancy among day (n = 5) and night (n = 3) workers by assessing their melatonin levels in a natural environment. Additionally, we describe the worker's metabolic profiles and compare the health status of the newborns between groups of day and night working mothers. Our results indicate an occurrence of assisted delivery (cesarean and forceps) among night workers. Moreover, the newborns of night workers showed lower Apgar index and breastfeeding difficulty indicating a worse condition to deal with the immediate outside the womb environment. Additionally, there was lower night-time melatonin production among pregnant night workers compared to day workers. These findings may be related to light-induced suppression of melatonin that occurs during night work. We conclude that night work and consequent exposure to light at unconventional times might compromise the success of pregnancy and the health of the newborn. Further studies need to be carried out to monitor pregnancy and newborn health in pregnant night workers.

25-Hydroxyvitamin D status, light exposure and sleep quality in UK dwelling South Asian and Caucasian postmenopausal women.

There is a lack of research into 25-hydroxyvitamin D (25(OH)D) status, light exposure and sleep patterns in South Asian populations. In addition, results of research studies are conflicting as to whether there is an association between 25(OH)D status and sleep quality. We investigated 25(OH)D status, self-reported and actigraphic sleep quality in n = 35 UK dwelling postmenopausal women (n = 13 South Asians, n = 22 Caucasians), who kept daily sleep diaries and wore wrist-worn actiwatch (AWL-L) devices for 14 days. A subset of n = 27 women (n = 11 South Asian and n = 16 Caucasian) also wore a neck-worn AWL-L device to measure their light exposure. For 25(OH)D concentration, South Asians had a median ± IQR of 43.8 ± 28.2 nmol/L, which was significantly lower than Caucasians (68.7 ± 37.4 nmol/L)(P = 0.001). Similarly, there was a higher sleep fragmentation in the South Asians (mean ± SD 36.9 ± 8.9) compared with the Caucasians (24.7 ± 7.1)(P = 0.002). Non-parametric circadian rhythm analysis of rest/activity patterns showed a higher night-time activity (L5) (22.6 ± 14.0 vs. 10.5 ± 4.4; P = 0.0008) and lower relative amplitude (0.85 ± 0.07 vs. 0.94 ± 0.02; P < 0.0001) in the South Asian compared with the Caucasian women. More South Asians (50%) met the criteria for sleep disorders (PSQI score >5) than did Caucasians (27%) (P = 0.001, Fishers Exact Test). However, there was no association between 25(OH)D concentration and any sleep parameter measured (P > 0.05) in either ethnic group. South Asians spent significantly less time in illuminance levels over 200 lx (P = 0.009) than did Caucasians. Overall, our results show that postmenopausal South Asian women have lower 25(OH)D concentration than Caucasian women. They also have higher sleep fragmentation, as well as a lower light exposure across the day. This may have detrimental implications for their general health and further research into sleep quality and light exposure in the South Asian ethnic group is warranted.

Diurnal preference, mood and the response to morning light in relation to polymorphisms in the human clock gene PER3.

PER3 gene polymorphisms have been associated with differences in human sleep-wake phenotypes, and sensitivity to light. The aims of this study were to assess: i) the frequency of allelic variants at two PER3 polymorphic sites (rs57875989 length polymorphism: PER3 4, PER3 5; rs228697 SNP: PER3 C, PER3 G) in relation to sleep-wake timing; ii) the effect of morning light on behavioural/circadian variables in PER3 4 /PER3 4 and PER3 5 /PER3 5 homozygotes. 786 Caucasian subjects living in Northern Italy donated buccal DNA and completed diurnal preference, sleep quality/timing and sleepiness/mood questionnaires. 19 PER3 4 /PER3 4 and 11 PER3 5 /PER3 5 homozygotes underwent morning light administration, whilst monitoring sleep-wake patterns and the urinary 6-sulphatoxymelatonin (aMT6s) rhythm. No significant relationship was observed between the length polymorphism and diurnal preference. By contrast, a significant association was observed between the PER3 G variant and morningness (OR = 2.10), and between the PER3 G-PER3 4 haplotype and morningness (OR = 2.19), for which a mechanistic hypothesis is suggested. No significant differences were observed in sleep timing/aMT6s rhythms between PER3 5 /PER3 5 and PER3 4 /PER3 4 subjects at baseline. After light administration, PER3 4 /PER3 4 subjects advanced their aMT6s acrophase (p < 0.05), and showed a trend of advanced sleep-wake timing. In conclusion, significant associations were observed between PER3 polymorphic variants/their combinations and both diurnal preference and the response to light.

Sleep and circadian rhythm disturbance in bipolar disorder.

BACKGROUND: Subjective reports of insomnia and hypersomnia are common in bipolar disorder (BD). It is unclear to what extent these relate to underlying circadian rhythm disturbance (CRD). In this study we aimed to objectively assess sleep and circadian rhythm in a cohort of patients with BD compared to matched controls. METHOD: Forty-six patients with BD and 42 controls had comprehensive sleep/circadian rhythm assessment with respiratory sleep studies, prolonged accelerometry over 3 weeks, sleep questionnaires and diaries, melatonin levels, alongside mood, psychosocial functioning and quality of life (QoL) questionnaires. RESULTS: Twenty-three (50%) patients with BD had abnormal sleep, of whom 12 (52%) had CRD and 29% had obstructive sleep apnoea. Patients with abnormal sleep had lower 24-h melatonin secretion compared to controls and patients with normal sleep. Abnormal sleep/CRD in BD was associated with impaired functioning and worse QoL. CONCLUSIONS: BD is associated with high rates of abnormal sleep and CRD. The association between these disorders, mood and functioning, and the direction of causality, warrants further investigation.

Clinical Decision Support: a 25 Year Retrospective and a 25 Year Vision.

OBJECTIVE: The objective of this review is to summarize the state of the art of clinical decision support (CDS) circa 1990, review progress in the 25 year interval from that time, and provide a vision of what CDS might look like 25 years hence, or circa 2040. METHOD: Informal review of the medical literature with iterative review and discussion among the authors to arrive at six axes (data, knowledge, inference, architecture and technology, implementation and integration, and users) to frame the review and discussion of selected barriers and facilitators to the effective use of CDS. RESULT: In each of the six axes, significant progress has been made. Key advances in structuring and encoding standardized data with an increased availability of data, development of knowledge bases for CDS, and improvement of capabilities to share knowledge artifacts, explosion of methods analyzing and inferring from clinical data, evolution of information technologies and architectures to facilitate the broad application of CDS, improvement of methods to implement CDS and integrate CDS into the clinical workflow, and increasing sophistication of the end-user, all have played a role in improving the effective use of CDS in healthcare delivery. CONCLUSION: CDS has evolved dramatically over the past 25 years and will likely evolve just as dramatically or more so over the next 25 years. Increasingly, the clinical encounter between a clinician and a patient will be supported by a wide variety of cognitive aides to support diagnosis, treatment, care-coordination, surveillance and prevention, and health maintenance or wellness.

Sleep patterns in Amazon rubber tappers with and without electric light at home.

Today's modern society is exposed to artificial electric lighting in addition to the natural light-dark cycle. Studies assessing the impact of electric light exposure on sleep and its relation to work hours are rare due to the ubiquitous presence of electricity. Here we report a unique study conducted in two phases in a homogenous group of rubber tappers living and working in a remote area of the Amazon forest, comparing those living without electric light (n = 243 in first phase; n = 25 in second phase) to those with electric light at home (n = 97 in first phase; n = 17 in second phase). Questionnaire data (Phase 1) revealed that rubber tappers with availability of electric light had significantly shorter sleep on work days (30 min/day less) than those without electric light. Analysis of the data from the Phase 2 sample showed a significant delay in the timing of melatonin onset in workers with electric light compared to those without electric light (p < 0.01). Electric lighting delayed sleep onset and reduced sleep duration during the work week and appears to interfere with alignment of the circadian timing system to the natural light/dark cycle.

Validation of an innovative method, based on tilt sensing, for the assessment of activity and body position.

Since there is less movement during sleep than during wake, the recording of body movements by actigraphy has been used to indirectly evaluate the sleep-wake cycle. In general, most actigraphic devices are placed on the wrist and their measures are based on acceleration detection. Here, we propose an alternative way of measuring actigraphy at the level of the arm for joint evaluation of activity and body position. This method analyzes the tilt of three axes, scoring activity as the cumulative change of degrees per minute with respect to the previous sampling, and measuring arm tilt for the body position inference. In this study, subjects (N = 13) went about their daily routine for 7 days, kept daily sleep logs, wore three ambulatory monitoring devices and collected sequential saliva samples during evenings for the measurement of dim light melatonin onset (DLMO). These devices measured motor activity (arm activity, AA) and body position (P) using the tilt sensing of the arm, with acceleration (wrist acceleration, WA) and skin temperature at wrist level (WT). Cosinor, Fourier and non-parametric rhythmic analyses were performed for the different variables, and the results were compared by the ANOVA test. Linear correlations were also performed between actimetry methods (AA and WA) and WT. The AA and WA suitability for circadian phase prediction and for evaluating the sleep-wake cycle was assessed by comparison with the DLMO and sleep logs, respectively. All correlations between rhythmic parameters obtained from AA and WA were highly significant. Only parameters related to activity levels, such as mesor, RA (relative amplitude), VL5 and VM10 (value for the 5 and 10 consecutive hours of minimum and maximum activity, respectively) showed significant differences between AA and WA records. However, when a correlation analysis was performed on the phase markers acrophase, mid-time for the 10 consecutive hours of highest (M10) and mid-time for the five consecutive hours of lowest activity (L5) with DLMO, all of them showed a significant correlation for AA (R = 0.607, p = 0.028; R = 0.582, p = 0.037; R = 0.620, p = 0.031, respectively), while for WA, only acrophase did (R = 0.621, p = 0.031). Regarding sleep detection, WA showed higher specificity than AA (0.95 ± 0.01 versus 0.86 ± 0.02), while the agreement rate and sensitivity were higher for AA (0.76 ± 0.02 versus 0.66 ± 0.02 and 0.71 ± 0.03 versus 0.53 ± 0.03, respectively). Cohen's kappa coefficient also presented the highest values for AA (0.49 ± 0.04) and AP (0.64 ± 0.04), followed by WT (0.45 ± 0.06) and WA (0.37 ± 0.04). The findings demonstrate that this alternative actigraphy method (AA), based on tilt sensing of the arm, can be used to reliably evaluate the activity and sleep-wake rhythm, since it presents a higher agreement rate and sensitivity for detecting sleep, at the same time allows the detection of body position and improves circadian phase assessment compared to the classical actigraphic method based on wrist acceleration.

Sleep and circadian rhythms in hospitalized patients with decompensated cirrhosis: effect of light therapy.

Patients with liver cirrhosis often exhibit sleep-wake abnormalities, which are, at least to some extent, circadian in origin. A relatively novel non-pharmacological approach to circadian disruption is appropriately timed bright light therapy. The aims of this pilot study were to investigate sleep-wake characteristics of a well-characterized population of inpatients with cirrhosis, and to evaluate the efficacy of bright light therapy in the hospital setting. Twelve consecutive inpatients with cirrhosis underwent complete sleep-wake assessment, to include qualitative and semi-quantitative (actigraphic) indices of night-time sleep quality, daytime sleepiness, diurnal preference, habitual sleep timing, quality of life, mood and circadian rhythmicity [i.e. urine collections for measurement of the melatonin metabolite 6-sulphatoxymelatonin (aMT6s)]. Patients showed extremely impaired night sleep quality (Pittsburg Sleep Quality Index global score: 16.3 ± 2.1) and daytime sleepiness was common (Epworth Sleepiness Scale: 8.3 ± 3.2). Five patients were randomly assigned to a single room in which lighting was controlled in relation to timing, spectral composition and intensity (lights on at 06:30 and off at 22:30, blue-enriched, more intense light in the morning, red-enriched, less intense light in the afternoon/evening); the others stayed in identical rooms with standard lighting. Sleep diaries revealed poor sleep quality, prolonged sleep latency (67 ± 138 min) and a reduced sleep efficiency (69 ± 21%). These features were confirmed by actigraphy (sleep efficiency: 71 ± 13%; fragmentation index: 55 ± 15%). Quality of life was globally impaired, and mood moderately depressed (Beck Depression Inventory: 19.4 ± 7.9). Seven patients underwent serial urine collections: no circadian aMT6s rhythm was detected in any of them, neither at baseline, nor during the course of hospitalization in either room (n = 4). In conclusion, sleep and circadian rhythms in hospitalized, decompensated patients with cirrhosis are extremely compromised. Treatment with bright light therapy did not show obvious, beneficial effects, most likely in relation to the severity of disturbance at baseline.

An open platform for personal health record apps with platform-level privacy protection.

One of the main barriers to the adoption of Personal Health Records (PHR) systems is their closed nature. It has been argued in the literature that this barrier can be overcome by introducing an open market of substitutable PHR apps. The requirements introduced by such an open market on the underlying platform have also been derived. In this paper, we argue that MyPHRMachines, a cloud-based PHR platform recently developed by the authors, satisfies these requirements better than its alternatives. The MyPHRMachines platform leverages Virtual Machines as flexible and secure execution sandboxes for health apps. MyPHRMachines does not prevent pushing hospital- or patient-generated data to one of its instances, nor does it prevent patients from sharing data with their trusted caregivers. External software developers have minimal barriers to contribute innovative apps to the platform, since apps are only required to avoid pushing patient data outside a MyPHRMachines cloud. We demonstrate the potential of MyPHRMachines by presenting two externally contributed apps. Both apps provide functionality going beyond the state-of-the-art in their application domain, while they did not require any specific MyPHRMachines platform extension.

Circadian phase assessment by ambulatory monitoring in humans: correlation with dim light melatonin onset.

The increased prevalence of circadian disruptions due to abnormal coupling between internal and external time makes the detection of circadian phase in humans by ambulatory recordings a compelling need. Here, we propose an accurate practical procedure to estimate circadian phase with the least possible burden for the subject, that is, without the restraints of a constant routine protocol or laboratory techniques such as melatonin quantification, both of which are standard procedures. In this validation study, subjects (N = 13) wore ambulatory monitoring devices, kept daily sleep diaries and went about their daily routine for 10 days. The devices measured skin temperature at wrist level (WT), motor activity and body position on the arm, and light exposure by means of a sensor placed on the chest. Dim light melatonin onset (DLMO) was used to compare and evaluate the accuracy of the ambulatory variables in assessing circadian phase. An evening increase in WT: WTOnset (WTOn) and "WT increase onset" (WTiO) was found to anticipate the evening increase in melatonin, while decreases in motor activity (Activity Offset or AcOff), body position (Position Offset (POff)), integrative TAP (a combination of WT, activity and body position) (TAPOffset or TAPOff) and an increase in declared sleep propensity were phase delayed with respect to DLMO. The phase markers obtained from subjective sleep (R = 0.811), WT (R = 0.756) and the composite variable TAP (R = 0.720) were highly and significantly correlated with DLMO. The findings strongly support a new method to calculate circadian phase based on WT (WTiO) that accurately predicts and shows a temporal association with DLMO. WTiO is especially recommended due to its simplicity and applicability to clinical use under conditions where knowing endogenous circadian phase is important, such as in cancer chronotherapy and light therapy.

An hour of bright white light in the early morning improves performance and advances sleep and circadian phase during the Antarctic winter.

Previous work has demonstrated that exposure to an hour of bright light in the morning and the evening during the Polar winter has beneficial effects on circadian phase. This study investigated the effect of a single hour of bright white morning light on circadian phase, sleep, alertness and cognitive performance. Nine individuals (eight male, one female, median age 30 years), wintering at Halley Research Station (75°S), Antarctica from 7th May until 6th August 2007, were exposed to bright white light for a fortnight from 08:30 to 09:30 h, with two fortnight control periods on either side. This sequence was performed twice, before and following Midwinter. Light exposure, sleep and alertness were assessed daily by actigraphy, sleep diaries and subjective visual analogue scales. Circadian phase (assessed by urinary 6-sulphatoxymelatonin rhythm) and cognitive performance were evaluated at the end of each fortnight. During light exposure circadian phase was advanced from 4.97 ± 0.96 decimal hours (dh) (mean ± SD) to 4.08 ± 0.68 dh (p = 0.003). Wake-up time was shifted by a similar margin from 8.45 ± 1.83 dh to 7.59 ± 0.78 dh (p < 0.001). Sleep start time was also advanced (p = 0.047) but by a lesser amount, consequently, actual sleep time was slightly reduced. There was no change in objective or subjective measures of sleep quality or subjective measures of alertness. An improvement in cognitive performance was found with both the Single Letter Cancellation Test (p < 0.001) and the Digit Symbol Substitution Test (p = 0.026) with preserved circadian variation. These beneficial effects of a single short duration light treatment may have implications not only for the Antarctic but other remote environments where access to natural light and delayed circadian phase, is problematic. These results require validation in larger studies at varying locations.

Comparison of Computer-based Clinical Decision Support Systems and Content for Diabetes Mellitus.

BACKGROUND: Computer-based clinical decision support (CDS) systems have been shown to improve quality of care and workflow efficiency, and health care reform legislation relies on electronic health records and CDS systems to improve the cost and quality of health care in the United States; however, the heterogeneity of CDS content and infrastructure of CDS systems across sites is not well known. OBJECTIVE: We aimed to determine the scope of CDS content in diabetes care at six sites, assess the capabilities of CDS in use at these sites, characterize the scope of CDS infrastructure at these sites, and determine how the sites use CDS beyond individual patient care in order to identify characteristics of CDS systems and content that have been successfully implemented in diabetes care. METHODS: We compared CDS systems in six collaborating sites of the Clinical Decision Support Consortium. We gathered CDS content on care for patients with diabetes mellitus and surveyed institutions on characteristics of their site, the infrastructure of CDS at these sites, and the capabilities of CDS at these sites. RESULTS: The approach to CDS and the characteristics of CDS content varied among sites. Some commonalities included providing customizability by role or user, applying sophisticated exclusion criteria, and using CDS automatically at the time of decision-making. Many messages were actionable recommendations. Most sites had monitoring rules (e.g. assessing hemoglobin A1c), but few had rules to diagnose diabetes or suggest specific treatments. All sites had numerous prevention rules including reminders for providing eye examinations, influenza vaccines, lipid screenings, nephropathy screenings, and pneumococcal vaccines. CONCLUSION: Computer-based CDS systems vary widely across sites in content and scope, but both institution-created and purchased systems had many similar features and functionality, such as integration of alerts and reminders into the decision-making workflow of the provider and providing messages that are actionable recommendations.

Benzo[c][2,7]naphthyridines as inhibitors of PDK-1.

A series of substituted benzo[c][2,7]-naphthyridines were prepared and showed good potency in inhibiting PDK-1. The synthesis and SAR of this series of compounds are presented as well as the X-ray crystal structure of one of these analogs in a complex with PDK-1.

Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.

4-Anilino-7-alkenylquinoline-3-carbonitriles as potent MEK1 kinase inhibitors.

A series of substituted 7-alkenyl 4[3-chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)]anilino-3-quinolinecarbonitrile analogs were synthesized and evaluated as MEK1 kinase inhibitors. The synthetic details, structure-activity relationships, biological activity, and selected oral exposure studies of these analogs are described. From these studies, compound 5m was chosen as a strong candidate for further evaluation. The selectivity of 5m was ascertained against a panel of 17 kinases, where activity was observed against EGFR, Src, Lyn, and IR kinases. Western blot studies in WM-266 cells demonstrated that 5m inhibited phosphorylation of ERK, while additional kinase pathways tested showed no inhibition at up to 10 microM of 5 m. PK studies, as well as a xenograft and in vivo biomarker studies are described for 5m.

Disturbances in melatonin and core body temperature circadian rhythms after minimal invasive surgery.

BACKGROUND: Sleep disturbances, fatigue and reduced general well-being frequently occur after minimal invasive surgery. The circadian rhythms of melatonin and core body temperature are central to the regulation of normal sleep. The aim of this study was to assess changes in these circadian rhythms after laparoscopic cholecystectomy. METHODS: Twelve women were studied before and after laparoscopic cholecystectomy. The major urinary melatonin metabolite, 6-sulphatoxymelatonin (aMT6s), and the core body temperature were measured for 1 day before and 1 day after surgery. The basal and maximum secretion of aMT6s were determined, as well as the timing and amplitude of aMT6s and the temperature rhythm. The patients' rest-activity and calculated sleep parameters were assessed by actigraphy. RESULTS: A significant delay in the timing of aMT6s rhythm was observed after surgery [median (range) peak time of aMT6s: after surgery, 05:49 h (02:57-08:23 h); before surgery, 04:32 h (02:18-06:49 h); P< or = 0.05]. The amplitude of the aMT6s rhythm was also significantly decreased after surgery [after surgery, 7.1 ng aMT6s/mg creatinine (1-15.9 ng); before surgery, 13.2 ng aMT6s/mg creatinine (2.9-22.7 ng); P< or = 0.005]. There was almost a 12-h phase delay of the core body temperature rhythm after surgery [peak time: before surgery, 17:39 h (15:17-22:06 h); after surgery, 05:14 h (03:24-21:43 h); P< or = 0.01]. CONCLUSIONS: Following laparoscopic cholecystectomy, there was a delay in the timing of the aMT6s rhythm and a decreased evening decline in the temperature rhythm.

Exogenous melatonin modifies rate of sexual maturation in domestic pullets.

Growing pullets were maintained on 14-h photoperiods and given diets supplemented with 25 mg of melatonin (MEL)/kg during the final 7 h of the photo-period to investigate the role of MEL in sexual development. Melatonin diets were fed to 70 d (to mimic a transfer from 7 to 14 h at 70 d), from 105 d onward (to mimic a transfer from 14 to 7 h at 105 d), or throughout the trial (to mimic constant 7-h photoperiods). Control birds, which were fed normal diets, were maintained on 7 or 14 h, transferred from 7 to 14 h at 70 d, or transferred from 14 to 7 h at 105 d. The MEL groups matured 6 to 11 d later than the constant 14-h controls. The group mimicking a transfer from 7 to 14 h matured 35 d later than photostimulated controls, the group mimicking a 14 to 7-h change at 105 d matured 41 d earlier than birds given a decrease in day length; the third group matured 13 d earlier than constant 7-h controls. Although these data suggest that the birds did not perceive the final 7 h of the photoperiod as being part of the night, when given MEL diets, residual plasma MEL during the first 7 h of the photoperiod was atypically high, possibly preventing an interpretation of day and night. However, continuously high plasma MEL did not result in birds responding as if in constant darkness, because birds transferred from darkness to 14 h at 70 d would not have matured at a similar time to birds changed from 14 h to darkness at 105 d. Plasma LH concentrations for birds mimicking a 7 to 14 h change at 70 d were not significantly different from constant 7-h controls after the transfer to normal diets. The later maturity of the experimental groups, compared with constant 14-h controls, clearly indicated that MEL had some influence over hypothalamic activity and gonadal development.

Keeping patients active and current in a patient portal.

Maintaining current contact information is crucial to the effectiveness of communication via patient portals. Patients who visit a portal infrequently, or whose contact information is not updated, may miss administrative notices or clinical messages from their doctor's office. We invited patients to review and update their contact information via a broadcast e-mail message sent to all patients in the patient portal, Patient Gateway. We report the effectiveness of the broadcast message approach to reaching registered patients.

Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.

Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE*) is a new development in protein detection for two-dimensional gels. Using mouse liver homogenates (control and paracetamol (N-acetyl-p-aminophenol, APAP)-treated), we have determined the quantitative variation in the 2-D DIGE process and established statistically valid thresholds for assigning quantitative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 fold for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labelling reaction. To minimise the thresholds, substantial user editing was required when using ImageMaster 2D-Elite software. The difference thresholds were applied to the test system and quantitative protein differences were determined using replicate gels of pool samples and single gels from multiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirrored almost without exception when the dye combination was reversed. Both pool and individual sample analyses provided unique data to the study. The inter-animal response variability in inbred mice was approximately nine times that contributed by the 2-D DIGE process. A number of the most frequently observed protein changes resulting from APAP-treatment were identified by mass spectrometry. Several of these can be rationalised based on available data on the mechanism of APAP hepatotoxicity but others cannot, indicating that proteomics can provide further insights into the biochemical basis of APAP toxicity.