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Abstract Background Discharge communication is essential to convey information regarding the care provided and follow-up plans after a visit to a hospital emergency department (ED), but it can be lacking for visits for pediatric mental health crises. Our objective was to co-design and conduct usability testing of new discharge communication interventions to improve pediatric mental health discharge communication. Methods The study was conducted in two phases using experience-based co-design (EBCD). In phase 1 (Sep 2021 to Jan 2022), five meetings were conducted with a team of six parents and two clinicians to co-design new ED discharge communication interventions for pediatric mental health care. Thematic analysis was used to identify patterns in team discussions and participant feedback related to discharge communication improvement and the Capability, Opportunity, Motivation, Behavior (COM-B) model was used to identify strategies to support the delivery of the new interventions. After meeting five, team members completed the Public and Patient Engagement Evaluation Tool (PPEET) to evaluate the co-design experience. In phase 2 (Apr to Jul 2022), intervention usability and satisfaction were evaluated by a new group of parents, youth aged 16–24 years, ED physicians, and nurses (n = 2 of each). Thematic analysis was used to identify usability issues and a validated 5-point Likert survey was used to evaluate user satisfaction. Evaluation results were used by the co-design team to finalize the interventions and delivery strategies. Results Two discharge communication interventions were created: a brochure for families and clinicians to use during the ED visit, and a text-messaging system for families after the visit. There was high satisfaction with engagement in phase 1 (overall mean PPEET score, 4.5/5). In phase 2, user satisfaction was high (mean clinician score, 4.4/5; mean caregiver/youth score, 4.1/5) with both interventions. Usability feedback included in the final intervention versions included instructions on intervention use and ensuring the text-messaging system activates within 12–24 h of discharge. Conclusions The interventions produced by this co-design initiative have the potential to address gaps in current discharge practices. Future testing is required to evaluate the impact on patients, caregivers, and health care system use after the ED visit.

Objective To construct a machine-learning (ML) model for health systems with organised falls prevention programmes to identify older adults at risk for fall-related admissions.Design This prognostic study used population-level administrative health data to develop an ML prediction model.Setting This study took place in Alberta, Canada during 2018–2019.Participants Albertans aged 65 and older with at least one prior admission. Those with palliative conditions or emigrated out of Alberta were excluded.Exposure Unit of analysis was the individual person.Main outcomes/measures We identified fall-related admissions. A CatBoost model was developed on 2018 data to predict risk of fall-related emergency department visits or hospitalisations. Temporal validation was done using 2019 data to evaluate model performance. We reported discrimination, calibration and other relevant metrics measured at the end of 2019 on both ranked predictions and predicted probability thresholds. A cost-savings simulation was performed using 2019 data.Results Final number of study participants was 224 445. The validation set had 203 584 participants with 19 389 fall-related events (9.5% pretest probability) and an ML model c-statistic of 0.70. The highest ranked predictions had post-test probabilities ranging from 40% to 50%. Net benefit analysis presented mixed results with some net benefit using the ML model in the 6%–30% range. The top 50 percentile of predicted risks represented nearly $C60 million in health system costs related to falls. Intervening on the top 25 or 50 percentiles of predicted risk could realise substantial (up to $C16 million) savings.Conclusion ML prediction models based on population-level administrative data can assist health systems with fall prevention programmes identify older adults at risk of fall-related admissions and reduce costs. ML predictions based on ranked predictions or probability thresholds could guide subsequent interventions to mitigate fall risks. Increased access to diverse forms of data could improve ML performance and further reduce costs.

Introduction: Hepcidin is a hormone that regulates systemic iron homeostasis. Serum hepcidin levels are under the influence of various stimuli, particularly inflammation and renal dysfunction. The measurement of hepcidin in circulation is a potentially useful clinical tool in the diagnosis, monitoring and treatment of iron metabolism disorder, although clinical interpretation of hepcidin level remains difficult. We evaluated he diagnostic potential and limitations of hepcidin-25 by investigating its relationship with iron and hematological indices, inflammation, and renal dysfunction. Methods: This retrospective study included 220 adult patients not requiring dialysis. Variations of biologically active hepcidin-25 were examined using a mass spectrometry-based assay in various inflammatory and renal states. The log[hepcidin]:log[ferritin] ratio was calculated as an hepcidin index. Results: In 220 adult patients not requiring dialysis, variation in hepcidin-25 level was significantly larger once CRP exceeded 10 mg/l (p 10 mg/l) and/or severe renal dysfunction (eGFR

ObjectivesTo describe the extent to which caregivers' emotional and communication needs were met during pediatric emergency department (PED) visits. Secondary objectives included describing the association of caregiver emotional needs, satisfaction with care, and comfort in caring for their child's illness at the time of discharge with demographic characteristics, caregiver experiences, and ED visit details.Study designElectronic surveys with medical record review were deployed at ten Canadian PEDs from October 2018 -March 2020. A convenience sample of families with children ResultsThis study recruited 2005 caregivers who self-identified as mothers (74.3%, 1462/1969); mean age was 37.8 years (SD 7.7). 71.7% (1081/1507) of caregivers felt their emotional needs were met. 86.4% (1293/1496) identified communication with the doctor as good/very good and 83.4% (1249/1498) with their child's nurse. Caregiver involvement in their child's care was reported as good/very good 85.6% (1271/1485) of the time. 81.8% (1074/1313) of caregivers felt comfortable in caring for their child at home at the time of discharge. Lower caregiver anxiety scores, caregiver involvement in their child's care, satisfactory updates, and having questions adequately addressed positively impacted caregiver emotional needs and increased caregiver comfort in caring for their child's illness at home.ConclusionApproximately 30% of caregivers presenting to PEDs have unmet emotional needs, over 15% had unmet communication needs, and 15% felt inadequately involved in their child's care. Family caregiver involvement in care and good communication from PED staff are key elements in improving overall patient experience and satisfaction.

Abstract Background Multiple evidence-based clinical practice guidelines (CPGs) exist to guide the management of concussion in children, but few have been translated into clinical pathways (CP), which operationalize guidelines into accessible and actionable algorithms that can be more readily implemented by health care providers. This study aimed to identify the clinical behaviours, attitudinal factors, and environmental contexts that potentially influence the implementation of a clinical pathway for pediatric concussion. Methods Semi-structured interviews were conducted from October 2017 to January 2018 with 42 emergency department clinicians (17 physicians, 25 nurses) at five urban emergency departments in Alberta, Canada. A Theoretical Domains Framework (TDF)-informed interview guide contained open-ended questions intended to gather feedback on the proposed pathway developed for the study, as well as factors that could potentially influence its implementation. Results The original 14 domains of the TDF were collapsed into 6 clusters based on significant overlap between domains in the issues discussed by clinicians: 1) knowledge, skills, and practice; 2) professional roles and identity; 3) attitudes, beliefs, and motivations; 4) goals and priorities; 5) local context and resources; and 6) engagement and collaboration. The 6 clusters identified in the interviews each reflect 2–4 predominant topics that can be condensed into six overarching themes regarding clinicians’ views on the implementation of a concussion CP: 1) standardization in the midst of evolving research; 2) clarifying and communicating goals; 3) knowledge dissemination and alignment of information; 4) a team-oriented approach; 5) site engagement; and 6) streamlining clinical processes. Conclusion Application of a comprehensive, evidence-based, and theory-driven framework in conjunction with an inductive thematic analysis approach enabled six themes to emerge as to how to successfullly implement a concussion CP.

Introduction Children and youth with mental health and addiction crises are a vulnerable patient group that often are brought to the hospital for emergency department care. We propose to evaluate the effect of a novel, acute care bundle that standardises a patient-centred approach to care.Methods and analysis Two paediatric emergency departments in Alberta, Canada are involved in this prospective, pragmatic, 29-month interventional quasi-experimental study. The acute care bundle comprises three components, applied when appropriate: (1) assessing self-harm risk at triage using the Ask Suicide-Screening Questionnaire (ASQ) to standardise the questions administered, enabling risk stratification; (2) use of the HEADS-ED (Home, Education, Activities/peers, Drug/alcohol, Suicidality, Emotions and behaviour, Discharge Resources) to focus mental health evaluations for those who screen high risk on the ASQ; and (3) implementation of a Choice And Partnership Approach to enable shared decision making in care following the emergency department visit. The overarching goal is to deliver the right care at the right place and time for the patients. The study design involves a longitudinal collection of data 12 months before and after the introduction of the bundle and the use of quality improvement strategies such as Plan-Do-Study-Act cycles during a 5-month run-in period to test and implement changes. The primary study end-point is child/youth well-being 1 month after the emergency department visit. Secondary outcomes include family functioning, child/youth well-being at 3 and 6 months, satisfaction with emergency department care, and health system outcomes (hospital admissions, length of emergency department stays, emergency department revisits).Ethics and dissemination The study is registered at www.ClinicalTrials.gov and has received ethics and operational approvals from study sites. The results of the study will be reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology statement. Results will be shared broadly with key policy and decision makers and disseminated in peer-reviewed academic journals and presentations at conferences.Trial registration number NCT04292379.

Diet is clearly implicated in the origin of colorectal cancer, with risk factors for the disease including reduced consumption of vegetables, fiber, and starch and increased consumption of red meat and animal fat. Several hypotheses have been developed to explain these associations. Most recently, McKeown-Eyssen and Giovannucci noted the similarity of the risk factors for colorectal cancer and those for insulin resistance and suggested that insulin resistance leads to colorectal cancer through the growth-promoting effect of elevated levels of insulin, glucose, or triglycerides. We briefly review the evidence from observational, epidemiological, and experimental animal studies linking diet with insulin resistance and colorectal cancer. The evidence suggests that diets high in energy and saturated fat and with high glycemic index carbohydrate and low levels of fiber and n-3 fatty acids lead to insulin resistance with hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. We then consider how insulin, the related insulin-like growth factors, triglycerides, and nonesterified fatty acids could lead to increased growth of colon cancer precursor lesions and the development of colorectal cancer. Finally, we consider the implications of this scheme on possible future research directions, including studies of satiety and clinical tests of the importance of insulin resistance in the colon carcinogenesis process. [ABSTRACT FROM AUTHOR]

McKeown-Eyssen (Cancer Epidemiol. Biomarkers Prevent., 3, 687-695, 1994) and Giovannucci (Cancer Causes Control, 6, 164-179, 1995), noting the strikingly similarity in lifestyle risk factors for colorectal cancer and insulin resistance, proposed that the hyperinsulinemia, glycemia and hypertriglyceridemia associated with insulin resistance promotes colon cancer. To compare the effect of diet on colon cancer promotion and insulin resistance in the F344 rat, we assessed the effect of fat, n-3 fatty acids and energy in pairwise comparisons on average size of aberrant crypt foci (ACF) and on glucose intolerance in the same animals in a single experiment. Diets high in fat and energy increased and diets with increased n-3 fatty acids and calorie restriction decreased both ACF growth and glucose intolerance compared with control diets. The measures of promotion of colon cancer and insulin resistance were strongly correlated (n = 98, r = 0.67, P < 0.001). In addition, both were highly correlated with daily energy intake (r = 0.62 and 0.66) and were also correlated with basal (post-prandial) insulin, glucose and triglycerides (r = 0.31-0.53, P > 0.01). We concluded that ACF growth and glucose intolerance are correlated for a wide range of diets and that increased circulating energy (glucose and triglycerides) may lead to both colon cancer promotion and insulin resistance. [ABSTRACT FROM PUBLISHER]

Background: Training in Bayesian reasoning may have limited impact on accuracy of probability estimates. In this study, our goal was to explore whether residents previously exposed to Bayesian reasoning use heuristics rather than Bayesian reasoning to estimate disease probabilities. We predicted that if residents use heuristics then post-test probability estimates would be increased by non-discriminating clinical features or a high anchor for a target condition. Method: We randomized 55 Internal Medicine residents to different versions of four clinical vignettes and asked them to estimate probabilities of target conditions. We manipulated the clinical data for each vignette to be consistent with either 1) using a representative heuristic, by adding non-discriminating prototypical clinical features of the target condition, or 2) using anchoring with adjustment heuristic, by providing a high or low anchor for the target condition. Results: When presented with additional non-discriminating data the odds of diagnosing the target condition were increased (odds ratio (OR) 2.83, 95% confidence interval [1.30, 6.15], p = 0.009). Similarly, the odds of diagnosing the target condition were increased when a high anchor preceded the vignette (OR 2.04, [1.09, 3.81], p = 0.025). Conclusions: Our findings suggest that despite previous exposure to the use of Bayesian reasoning, residents use heuristics, such as the representative heuristic and anchoring with adjustment, to estimate probabilities. Potential reasons for attribute substitution include the relative cognitive ease of heuristics vs. Bayesian reasoning or perhaps residents in their clinical practice use gist traces rather than precise probability estimates when diagnosing.

Abstract Introduction Physicians experience consistent environmental pressures while doing thier work. To give students practice making clinical decisions in a time- and cost-limited scenario, we created this group learning, case-based activity for first-year medical students during their second semester. During this activity students are tasked with diagnosing a pediatric patient who presents with traveler's diarrhea, but also signs of an underlying chronic anemia. Methods This 2-hour session began with students reviewing the case in groups of 8 for 15 minutes after which time they were allowed to purchase physical exam results, and later test results from a simulated lab. However, all student groups had a limited number of tokens which restricts what information they could buy. After a set time, students presented their diagnosis and there was a facilitated discussion. The exercise was followed by 5–6 NBME-style multiple choice questions on the basic science related to this case. Each question was presented and student groups had 60–90 seconds to respond via an electronic audience response system. To evaluate the session, students were given an IRB-approved, Likert scale questionnaire via Survey Monkey. Results This type of exercise was originally developed for our scholars, which includes 150 students. However, we have run such cases also with the entire class of 500 students. Even though the preparation is more laborious than a typical group learning exercise, faculty find this fun to do. Students, too, like this format; the majority think the additional simulated pressure is more helpful than normal clinical cases. Discussion This type of exercise has also been used for remedial work with students. Lab-token exercises may also be appropriate in the training of other health care workers (e.g., nurse practitioners).

Background: Ultrasonography is increasingly used for teaching physical examination in medical schools. This study seeks the opinions of educators as to which physical examinations would be most enhanced by the addition of ultrasonography. We also asked when ultrasound-aided physical examination teaching could have deleterious effects if used outside its intended scope. Methods: All of the educators from the University of Calgary Master Teacher Program were invited to complete a 22-item paper-based survey. Survey items were generated independently by two investigators, with input from an expert panel (N = 5). Results: Of the 36 educators, 27 (75%) completed the survey. Examinations identified to be potentially most useful included: measuring the size of the abdominal aorta, identifying the presence/absence of ascites, identifying the presence/absence of pleural effusions, and measuring the size of the bladder. Examinations thought to be potentially most harmful included: identifying the presence/absence of intrauterine pregnancy, measuring the size of the abdominal aorta, and identifying the presence/absence of pericardial effusion. Conclusions: Examinations that are potentially the most useful may also be potentially the most harmful. When initiating an ultrasound curriculum for physical examinations, educators should weigh the risks and benefits of examinations chosen.

Background: Most incorrect diagnoses involve at least one cognitive error, of which premature closure is the most prevalent. While metacognitive strategies can mitigate premature closure in inexperienced learners, these are rarely studied in experienced physicians. Our objective here was to evaluate the effect of analytic information processing on diagnostic performance of nephrologists and nephrology residents. Methods: We asked nine nephrologists and six nephrology residents at the University of Calgary and Glasgow University to diagnose ten nephrology cases. We provided presenting features along with contextual information, after which we asked for an initial diagnosis. We then primed participants to use either hypothetico-deductive reasoning or scheme-inductive reasoning to analyze the remaining case data and generate a final diagnosis. Results: After analyzing initial hypotheses, both nephrologists and residents improved the accuracy of final diagnoses (31.1% vs. 65.6%, p < 0.001, and 40.0% vs. 70.0%, p < 0.001, respectively). We found a significant interaction between experience and analytic processing strategy (p = 0.002): nephrology residents had significantly increased odds of diagnostic success when using scheme-inductive reasoning (odds ratio [95% confidence interval] 5.69 [1.59, 20.33], p = 0.007), whereas the performance of experienced nephrologists did not differ between strategies (odds ratio 0.57 [0.23, 1.39], p = 0.2). Discussion: Experienced nephrologists and nephrology residents can improve their performance by analyzing initial diagnostic hypotheses. The explanation of the interaction between experience and the effect of different reasoning strategies is unclear, but may relate to preferences in reasoning strategy, or the changes in knowledge structure with experience.

Background: This pilot study compared performance of University of Calgary students on internal clerkship examinations with corresponding National Board of Medical Examiners (NBME) subject examinations. Methods: Between April and October 2007, students completed internal and NBME subject examinations following six mandatory rotations. Local faculty within each discipline set the minimum performance level (MPL) for internal examinations. Two methods of standard setting were considered for NBME exams and a sensitivity analysis was performed. Corresponding internal and NBME examination scores were compared using McNemar’s discordant pair analysis. Results: A significant and unexpected difference in failure rate between internal and external examinations was found in all clerkships. 1.4% of students were below the MPL for internal examinations and 27.3% (modified Angoff) or 25.9% (mean Hofstee compromise) (p

Background: Medical students encounter a variety of experiences that have an impact on their emerging professional identity. Clerkship, in particular, presents opportunities for students to consider their career options and decide upon a career path. The process of developing their professional identity begins well before clerkship, however. Anecdotal evidence suggests that interests in medicine begin as early as childhood. This study retrospectively examines the decision-making process clerks make in choosing medicine as a career. Methods: A total of 76 clerks (36 male, 34 female, 6 not reported) responded to four open-ended and two follow-up questions that measure career interests and pursuits. Questions addressed when and how students developed interests in medicine and alternate careers before beginning medical school. An additional eight closed questions drawn from the Ego Status Extended Objective Measure of Ego Identity Status II (EOM-EIS-II) were administered. Content analyses and inter-rater reliability analyses were conducted to classify students according to Marcia’s1 four ego identity statuses. Results: Having obtained high inter-rater consistency (Cohen’s Kappa coefficient of 0.92), responses to the open-ended questions resulted in the classification of three identity statuses. In total, 49.3% of students were in the ‘achieved’ (high exploration and commitment to choices) status and 48.1% were in the ‘foreclosed’ (low exploration but high commitment to choices) status. A small percentage (1.3%) of students were in the ‘moratorium’category (high exploration but low commitment to choices), while none of the students were in the ‘diffused’ (low exploration and low commitment to choices) category. Conclusions: With approximately half of the students demonstrating a ‘foreclosed’ status, this study reveals that despite exposure to a variety of careers when attending university, only half of the students had seriously pursued a career outside of medicine. The majority of students, moreover, developed an interest in medicine before adulthood, and did so independently from parental influence.

Abstract This team-based learning (TBL) module encourages learners to work effectively in small groups to discuss and understand the normal anatomy and physiology of a cell, and to be able to describe which abnormal cellular processes produce a specific disease, outlined in the clinical application exercise. The learners will also discuss the anatomy and physiology of nerve transmission, and relate the underlying mechanisms of this to the disease in the application exercise. Finally, learners will determine the inheritance pattern of the disease in the application exercise, and use this for risk calculations while applying the Hardy-Weinberg principle of population genetics. The module follows the basic TBL structure and is written to utilize the “Four S's” in order to foster teamwork and group discussion. This TBL session requires approximately two hours to complete and includes an individual readiness assessment test, a group readiness assessment test, and a group clinical application case. This module allowed first-year students to review several common basic science concepts in a clinical setting. The most effective part of the case was the inclusion of lab data that helped the student to develop their analytical skills to come to some conclusions about the patient, but not necessarily a diagnosis. This stimulated great discussion and helped the students see the relevance of basic science education in the context of medicine. The student feedback from this module was all positive.

ABSTRACT Background: Medical student rotations in community practice settings are increasingly common within pediatric clerkship curricula yet little evidence exists to support the quality of the educational exposure. Purpose: To assess the impact of clerkship site (community setting vs. exposure to an Academic Health Sciences Center) on the following educational outcomes: 1. Clinical Performance; 2. Examination Performance; 3. Written Assignment Performance; and 4. Successful Matching to a Canadian Pediatric Residency Program. Methods: 340 medical students from the graduating classes of 2007 and 2008 at the University of Toronto, Canada were studied. Rotation performance (clinical assessment, examination mark, and written assignment mark) and acceptance into a Canadian pediatric residency program were assessed in relation to clerkship rotation site. These outcomes were assessed while controlling for the following potential confounders: 1) Pre-clerkship career preference and 2) Pre-rotation site preference as expressed by each medical student. Results: 172 medical students completed rotations that included exposure to an academic health sciences center, while 168 medical students had exclusive exposure to the community setting. Students who completed exclusively community-based pediatric rotations received slightly higher clinical evaluations (p=0.006), but not exam marks (p=0.812) nor written assignment marks (p=0.086). Students who had expressed an interest in paediatrics as a career prior to beginning their clerkship performed better during paediatric clerkship regardless of site (p= .0003) and were more likely to choose a clerkship setting that included exposure to an Academic Health Sciences Center (p=.052). Clerkship setting was not found to impact on successful matching to a Canadian pediatric residency program (p=0.171). Discussion: These results help support the decision of curriculum committees to incorporate the use of community practice settings and inform students and faculty as to the validity of distributed medical education within the field of pediatric medical education.

PhotoCD is a novel electronic format which uses digital technology to scan colour or monochrome photographs, store them on compact discs, and then display them as highquality images on a TV or on a monitor screen. A technical description of this technology can be found in Beisner (1993), Bowers (1994) and Chen (1993). Supporting software is available to access and manipulate the images for use in interactive learning.

En una granja con una población aproximada de 2000 vientres se presentó un brote de Gastroenteritis Transmisible de los Cerdos (GTC) en 1977. Desde esa época en la granja se han presentado diarreas constantes en lechones, por lo que se sospechó que se tra

Background: Chronic inflammation and oxidative stress are thought to be involved in colorectal cancer development. These processes may contribute to leakage of bacterial products, such as lipopolysaccharide (LPS) and flagellin, across the gut barrier. The objective of this study, nested within a prospective cohort, was to examine associations between circulating LPS and flagellin serum antibody levels and colorectal cancer risk., Methods: A total of 1,065 incident colorectal cancer cases (colon, n = 667; rectal, n = 398) were matched (1:1) to control subjects. Serum flagellin- and LPS-specific IgA and IgG levels were quantitated by ELISA. Multivariable conditional logistic regression models were used to calculate ORs and 95% confidence intervals (CI), adjusting for multiple relevant confouding factors., Results: Overall, elevated anti-LPS and anti-flagellin biomarker levels were not associated with colorectal cancer risk. After testing potential interactions by various factors relevant for colorectal cancer risk and anti-LPS and anti-flagellin, sex was identified as a statistically significant interaction factor (Pinteraction < 0.05 for all the biomarkers). Analyses stratified by sex showed a statistically significant positive colorectal cancer risk association for men (fully-adjusted OR for highest vs. lowest quartile for total anti-LPS + flagellin, 1.66; 95% CI, 1.10-2.51; Ptrend, 0.049), whereas a borderline statistically significant inverse association was observed for women (fully-adjusted OR, 0.70; 95% CI, 0.47-1.02; Ptrend, 0.18)., Conclusion: In this prospective study on European populations, we found bacterial exposure levels to be positively associated to colorectal cancer risk among men, whereas in women, a possible inverse association may exist., Impact: Further studies are warranted to better clarify these preliminary observations., (©2016 American Association for Cancer Research.)

Epidemiological studies have demonstrated clear associations between specific dietary and environmental risk factors and incidence of colorectal cancer, but the mechanisms responsible for these associations are not known. An animal model could facilitate such an understanding. Both genotoxic and nongenotoxic carcinogens induce aberrant crypt foci (ACF) in the colons of F344 rats. F344 rats were provided with diets that contained putative risk factors for CRC: low calcium and low vitamin D, high iron, high fructose, and decreased light (UV) exposure or a control diet for 14 wk. The rats were then assessed with biochemical measures and by topological examination for evidence of colon abnormalities. Circulating ionized calcium was decreased from 2.85 to 1.69 mmol/L, and ACF were increased from 0.7 to 13.6 lesions/colon (both P < 0.001). Rats exposed to the multiple environmental conditions associated with colon cancer, developed ACF similar to the heterogeneous or ill-defined ACF in the human colon. Heterogeneous ACF are the most frequently seen in humans and are also seen in rats shortly after exposure to the non-genotoxic colon carcinogen, dextransulfate sodium. The rodent model could be used to assess the pathways from diet and environment to colon cancer and to provide guidance for clinical studies.

Background: A large proportion of colorectal cancers are thought to be associated with unhealthy dietary and lifestyle exposures, particularly energy excess, obesity, hyperinsulinemia, and hyperglycemia. It has been suggested that these processes stimulate the production of toxic reactive carbonyls from sugars such as glyceraldehyde. Glyceraldehyde contributes to the production of a group of compounds known as glyceraldehyde-derived advanced glycation end-products (glycer-AGEs), which may promote colorectal cancer through their proinflammatory and pro-oxidative properties. The objective of this study nested within a prospective cohort was to explore the association of circulating glycer-AGEs with risk of colorectal cancer., Methods: A total of 1,055 colorectal cancer cases (colon n = 659; rectal n = 396) were matchced (1:1) to control subjects. Circulating glycer-AGEs were measured by a competitive ELISA. Multivariable conditional logistic regression models were used to calculate ORs and 95% confidence intervals (95% CI), adjusting for potential confounding factors, including smoking, alcohol, physical activity, body mass index, and diabetes status., Results: Elevated glycer-AGEs levels were not associated with colorectal cancer risk (highest vs. lowest quartile, 1.10; 95% CI, 0.82-1.49). Subgroup analyses showed possible divergence by anatomical subsites (OR for colon cancer, 0.83; 95% CI, 0.57-1.22; OR for rectal cancer, 1.90; 95% CI, 1.14-3.19; Pheterogeneity = 0.14)., Conclusions: In this prospective study, circulating glycer-AGEs were not associated with risk of colon cancer, but showed a positive association with the risk of rectal cancer., Impact: Further research is needed to clarify the role of toxic products of carbohydrate metabolism and energy excess in colorectal cancer development., (©2015 American Association for Cancer Research.)

Previously, we showed that dietary fructose or its carbonyl metabolites, glyceraldehyde and glycolaldehyde, could be oxidized by inflammatory reactive oxygen species (ROS), products of immune cells, to form highly toxic and genotoxic products, such as glyoxal. Glycolaldehyde-caused hepatocyte protein carbonylation likely resulted from glyoxal, an autoxidation product formed by ROS. Although hepatocyte protein carbonylation by glyoxal or d-glycolaldehyde was rapid, the product was unstable. Glyceraldehyde-induced protein carbonylation was slower and was also less cytotoxic. Non-toxic concentrations of H(2)O(2) were then used to mimic inflammation and oxidative stress associated with fructose-induced non-alcoholic steatohepatitis (NASH). A slow infusion of H(2)O(2) markedly increased glyoxal, glyceraldehyde, and glycolaldehyde-induced cytotoxicity and protein carbonylation. However, it had a smaller effect on glyceraldehyde-induced protein carbonylation. The cytotoxicities of both aldehydes were increased if glutathione (GSH)-depleted hepatocytes were used, presumably because of the increased ROS formation and subsequent glyoxal-induced protein carbonylation. Catalytic amounts of Cu or Fe increased the glycolaldehyde and glyceraldehyde-induced cytotoxicity and protein carbonylation resulting from autoxidation to glyoxal. Glyceraldehyde and glycolaldehyde were also detoxified by mitochondrial aldehyde dehydrogenase (ALDH2) as ALDH2 inhibitors increased their cytotoxicity. Hydroxypyruvate has not been previously tested for toxicity and was found to be the most toxic fructose metabolite. Catalytic amounts of Cu or Fe caused hydroxypruvate autoxidation, which formed extensive ROS, glycolaldehyde and glyoxal. Iron chelators EGTA or deferoxamine inhibited cytotoxicity as well as the extensive ROS formation. The Girard assay confirmed that glyoxal was a common autoxidation product from glyceraldehyde, glycolaldehyde and hydroxypyruvate., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)

Chronic hyperglycemia in diabetic patients often leads to chronic side effects associated with protein glycation and the formation of reactive carbonyl species, such as methylglyoxal (MGO) and glyoxal (GO). We have shown that both MGO and GO carbonylated bovine serum albumin (BSA) in vitro to the same degree and stability. The carbonylated BSA formed initially could be a reversible Schiff base as the UV absorbance formed after the addition of 2,4-dinitrophenylhydrazine was decreased when sodium borohydride was added. MGO and GO also carbonylated hepatocyte protein rapidly with similar dose and time dependence. In contrast to BSA carbonylation, the amount of carbonylated proteins in hepatocytes decreased over time, much more rapidly for hepatocytes treated with MGO than with GO. This could be attributed to the rapid hepatocyte metabolism of MGO with glyoxalase I, the predominant detoxification enzyme for MGO. Protein carbonylation and the associated toxicity caused by GO and MGO were studied in the following hepatocyte models: (1) control hepatocytes, (2) glutathione (GSH)-depleted hepatocytes, (3) mitochondrial aldehyde dehydrogenase (ALDH2)-inhibited hepatocytes, (4) hepatocyte inflammation model, and (5) catalase-inhibited hepatocyte model. Carbonylation and cytotoxicity caused by MGO or GO was markedly increased in GSH-depleted hepatocytes as compared to control hepatocytes. Hepatocytes exposed to non-toxic concentrations of H(2)O(2) or hepatocytes treated with catalase inhibitors also showed a marked increase in GO-caused cytotoxicity and protein carbonylation, whereas there were only minor increases with MGO. The GO effect was attributed to potential radical formation and the inhibition effect of H(2)O(2) on aldehyde dehydrogenase, a major GO metabolising enzyme. GO-caused cytotoxicity and protein carbonylation were also increased with ALDH2-inhibited hepatocytes whereas such an increase was only observed with MGO in GSH-depleted hepatocytes., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)

Insulin resistance and hepatotoxicity induced in high fructose fed rats may involve fructose derived endogenous toxins formed by inflammation. Thus fructose was seventy-fold more toxic if hepatocytes were exposed to non-toxic levels of hydrogen peroxide (H(2)O(2)) released by inflammatory cells. This was prevented by iron (Fe) chelators, hydroxyl radical scavengers, and increased by Fe, copper (Cu) or catalase inhibition. Fructose or glyceraldehyde/dihydroxyacetone metabolites were oxidized by Fenton radicals to glyoxal. Glyoxal (15 microM) cytotoxicity was increased about 200-fold by H(2)O(2). Glycolaldehyde was enzymically formed from glyceraldehyde, the fructokinase/aldolase B product of fructose. Glycolaldehyde cytotoxicity was increased 20-fold by H(2)O(2). The oxidative stress cytotoxicity induced was attributed to the Fenton oxidation of glycolaldehyde forming glycolaldehyde radicals and glyoxal, since cytotoxicity was prevented by aminoguanidine (glyoxal trap) or Fenton inhibitors. Glyoxal was also the Fenton product responsible for glycolaldehyde protein carbonylation as carbonylation was prevented by aminoguanidine or Fenton inhibitors.

Dietary fructose consumption is one of the environmental factors contributing to the development of obesity and a fatty liver (hepatic steatosis). A two-hit hypothesis has been proposed for progression of hepatic steatosis to the more serious non-alcoholic steatosis (NASH), with the first hit being hepatic steatosis, and the second hit being inflammation and associated oxidative stress caused by reactive oxygen species (ROS) formation. As well, fructose-fed rats develop insulin resistance and serum levels of methylglyoxal, a glycolytic metabolite, are increased. Previously we reported that glyoxal-induced hepatocyte cytotoxicity could be attributed to mitochondrial toxicity as mitochondrial membrane potential was decreased and cytotoxicity was increased several orders of magnitude by low non-cytotoxic doses of H(2)O(2) (hepatocyte inflammation model). In this study, we have assessed the toxicity of fructose towards hepatocytes and investigated the molecular cytotoxic mechanisms involved. Fructose itself was only toxic at 1.5M, whereas 12 mM caused 50% cell death in 2h if the hepatocytes were exposed to a non-cytotoxic dose of H(2)O(2) continuously generated by glucose and glucose oxidase. The cytotoxic mechanism involved oxidative stress as ROS and H(2)O(2) formation preceded cytotoxicity, and cytotoxicity was prevented by radical scavengers, lipid antioxidants and ROS scavengers. It is proposed that the highly potent Fenton derived ROS catalyse the oxidation of fructose and particularly its carbonyl metabolites glycolaldehyde, dihydroxyacetone, glyceraldehyde. The carbon radicals and glyoxal formed compromise the cell's resistance to H(2)O(2).

Thermolysis of proteins produces xenobiotic amino-acids such as the potentially toxic lysinoalanine, and the alkylating agent, dehydroalanine, which have been considered possible health hazards. We observed that thermolyzed casein promoted aberrant crypt foci (ACF) and colon cancer growth in rats initiated with azoxymethane and speculated that promotion might be due to the formation of these compounds. To test this notion we first measured the concentration of the modified amino acids as a function of thermolysis time. The concentration of dehydroalanine in the casein paralleled the degree of promotion, that of lysinoalanine did not. We then tested diets containing foods with high levels of dehydroalanine (thermolyzed sodium-caseinate, cooked Swiss cheese) for their effect on ACF promotion. They decreased the number and/or size of ACF significantly, indicating that dehydroalanine did not promote, but protected rats against colon carcinogenesis. These results do not support the notion that lysinoalanine or dehydroalanine are a hazard with respect to colon carcinogenicity.

A thermolyzed diet has the potential of providing exogenous oxidative stress in the form of advanced glycation end-products (AGE) and decreased thiamin. There is then a possibility that it could result in intracellular exposure to alpha-oxoaldehydes (glyoxal and methylglyoxal (MG)) with metabolic and genetic consequences. Two groups of Fischer 344 rats were fed the following diets: group A was given an AIN93G diet (control diet), while group B was given a thermolyzed AIN93G diet for 77 days. At the end of 77 days TK activity in red blood cells; glyoxal/MG levels in the plasma; glyoxal/MG HI protein adducts and dicarbonyls in the plasma, liver and colon tissues; glutathione levels of whole blood; and oxidative stress/inflammatory markers in the colon were measured. The thermolyzed diet resulted in: decreased thiamin status, increased plasma levels of glyoxal/MG and their adducts, increased protein dicarbonyls in the liver and plasma, lowered blood glutathione levels, increased infiltration of macrophages and increased colon nitrotyrosine levels. The thermolyzed diet increased the body burden of AGEs and decreased the thiamin status of the rats. This increased endogenous alpha-oxoaldehydes and oxidative stress has the potential to injure tissues that have low levels of antioxidant defenses such as the colon.

The B vitamins are water-soluble vitamins required as coenzymes for enzymes essential for cell function. This review focuses on their essential role in maintaining mitochondrial function and on how mitochondria are compromised by a deficiency of any B vitamin. Thiamin (B1) is essential for the oxidative decarboxylation of the multienzyme branched-chain ketoacid dehydrogenase complexes of the citric acid cycle. Riboflavin (B2) is required for the flavoenzymes of the respiratory chain, while NADH is synthesized from niacin (B3) and is required to supply protons for oxidative phosphorylation. Pantothenic acid (B5) is required for coenzyme A formation and is also essential for alpha-ketoglutarate and pyruvate dehydrogenase complexes as well as fatty acid oxidation. Biotin (B7) is the coenzyme of decarboxylases required for gluconeogenesis and fatty acid oxidation. Pyridoxal (B6), folate and cobalamin (B12) properties are reviewed elsewhere in this issue. The experimental animal and clinical evidence that vitamin B therapy alleviates B deficiency symptoms and prevents mitochondrial toxicity is also reviewed. The effectiveness of B vitamins as antioxidants preventing oxidative stress toxicity is also reviewed.

The B vitamins are water-soluble vitamins that are required as coenzymes for reactions essential for cellular function. This review focuses on the essential role of vitamins in maintaining the one-carbon transfer cycles. Folate and choline are believed to be central methyl donors required for mitochondrial protein and nucleic acid synthesis through their active forms, 5-methyltetrahydrofolate and betaine, respectively. Cobalamin (B12) may assist methyltetrahydrofolate in the synthesis of methionine, a cysteine source for glutathione biosynthesis. Pyridoxal, pyridoxine and pyridoxamine (B6) seem to be involved in the regeneration of tetrahydrofolate into the active methyl-bearing form and in glutathione biosynthesis from homocysteine. Other roles of these vitamins that are relevant to mitochondrial functions will also be discussed. However these roles for B vitamins in cell function are mostly theoretically based and still require verification at the cellular level. For instance it is still not known what B vitamins are depleted by xenobiotic toxins or which cellular targets, metabolic pathways or molecular toxic mechanisms are prevented by B vitamins. This review covers the current state of knowledge and suggests where this research field is heading so as to better understand the role vitamin Bs play in cellular function and intermediary metabolism as well as molecular, cellular and clinical consequences of vitamin deficiency. The current experimental and clinical evidence that supplementation alleviates deficiency symptoms as well as the effectiveness of vitamins as antioxidants will also be reviewed.

The similarity in risk factors for insulin resistance and colorectal cancer (CRC) led to the hypothesis that markers of insulin resistance, such as elevated circulating levels of insulin, glucose, fatty acids, and triglycerides, are energy sources and growth factors in the development of CRC. The objective was thus to examine the individual and combined effects of these circulating factors on colorectal epithelial proliferation in vivo. Rats were fasted overnight, randomized to six groups, infused iv with insulin, glucose, and/or Intralipid for 10 h, and assessed for 5-bromo-2-deoxyuridine labeling of replicating DNA in colorectal epithelial cells. Intravenous infusion of insulin, during a 10-h euglycemic clamp, increased colorectal epithelial proliferation in a dose-dependent manner. The addition of hyperglycemia to hyperinsulinemia did not further increase proliferation. Intralipid infusion alone did not affect proliferation; however, the combination of insulin, glucose, and Intralipid infusion resulted in greater hyperinsulinemia than the infusion of insulin alone and further increased proliferation. Insulin infusion during a 10-h euglycemic clamp decreased total IGF-I levels and did not affect insulin sensitivity. These results provide evidence for an acute role of insulin, at levels observed in insulin resistance, in the proliferation of colorectal epithelial cells in vivo.

We hypothesized that in marginal thiamin deficiency intracellular alpha-oxoaldehydes form macromolecular adducts that could possibly be genotoxic in colon cells; and that in the presence of oxidative stress these effects are augmented because of decreased detoxification of these aldehydes. We have demonstrated that reduced dietary thiamin in F344 rats decreased transketolase activity and increased alpha-oxoaldehyde adduct levels. The methylglyoxal protein adduct level was not affected by oral glyoxal or methylglyoxal in the animals receiving thiamin at the control levels but was markedly increased in the animals on a thiamin-reduced diet. These observations are consistent with our suggestion that the induction of aberrant crypt foci with marginally thiamin-deficient diets may be a consequence of the formation of methylglyoxal adducts.

Colorectal cancer risk is associated with biochemical markers for B-vitamin deficiency, insulin resistance and colonic inflammation, suggesting that these three conditions are each involved in colon carcinogenesis. We expected that dietary supplements of folic acid, n-3 fatty acids and calcium would reduce the markers and thus possibly cancer risk. We therefore randomised 98 participants, with previous colonic polyps or intramucosal carcinomas, to a combined treatment of supplementary folic acid, fish oil and calcium carbonate, or placebos for 28 days. Blood and faecal samples were obtained prior to and at the conclusion of the intervention and analysed for plasma folate, homocysteine, insulin, free fatty acids, triglycerides and faecal calprotectin. In addition, plasma vitamin B12, thiamin, glucose and C-reactive protein were assessed. Our supplemental strategy modestly affected some of the biomarkers associated with folate metabolism and insulin resistance, but had no effect on those associated with colonic inflammation. This pilot study demonstrates the feasibility and practicality of clinical trials aimed at reducing diet-related biochemical risk markers for colon cancer. We suggest that long-term intervention studies with tumour-related end points should be undertaken when the intervention agents used are found effective in short-term biochemical risk marker trials.

Polyethylene glycol 8000 inhibits the formation of tumors and of aberrant crypt foci (ACF) in carcinogen-initiated rats. We asked: is the inhibition associated with a reduction of colonic inflammation and an increase in colonic cell permeability? Twenty-eight, male F 344 rats were divided into two groups, 10 control animals and 18 animals initiated with azoxymethane. Nine of the rats in the carcinogen-initiated group were given a diet with 5% PEG 8000 in an AIN-93 based, high fat diet. The other nine, and the control group received the diet without the addition of PEG. Nine weeks later, the rats receiving the diet containing PEG had a 43% reduction in ACF (P<0.001) compared with the carcinogen-initiated rats on the control diet, a result confirming earlier observations that PEG inhibits colon carcinogenesis. The animals receiving the diet containing PEG also had a 10-fold reduction in fecal granulocyte marker protein (GMP) (P<0.001) compared with both the carcinogen-treated and the control animals. PEG reduced inflammation below the levels of carcinogen-treated and of untreated animals. Fecal water from the rats receiving PEG did not reduce transepithelial resistance of, or manitol flux through, human Caco-cells grown as monolayers in vitro. PEG may reduce colon carcinogenesis through a mechanism involving colonic inflammation.

Thiamin deficiency leads to the endogenous formation of genotoxic alpha-oxoaldehydes (glyoxals). To evaluate whether marginal deficiency poses a carcinogenesis risk we fed rats AIN-76A sucrose-based diets containing thiamin at 4.9 (control), 1.6 or 1.0 mg/kg diet and examined their colons after 160 days. Reduced thiamin increased aberrant crypt foci (ACF) from 1.14+/-0.46 to 3.70+/-1.17 and 2.60+/-1.02 ACF/colon in the absence of exogenous carcinogen or of symptoms of beriberi. Since typical Western diets can provide marginal levels of thiamin with high levels of simple sugars, individuals could be exposed to an increased risk of colon and perhaps other cancers.

Glyoxals are reactive alpha-oxoaldehydes that are formed endogenously from sugars, the levels of which are increased in various pathological conditions associated with hyperglycaemia and thiamine deficiency. However, the molecular cytotoxic mechanisms of glyoxal are not known. Results presented here and in the other studies cited provide a glimpse into the cytotoxicity mechanisms involved and their pathological implications. We found that glyoxal (10 microM) markedly increased the susceptibility of hepatocyte glutathione (GSH) to oxidation by hydrogen peroxide (H(2)O(2)) and markedly increased cytotoxicity by compromising the cellular antioxidant enzyme system. At higher concentrations, glyoxal was cytotoxic towards hepatocytes, which can be attributed to GSH depletion, oxidative stress and mitochondrial toxicity. Aminoguanidine or penicillamine protected the hepatocytes. Glyoxal cytotoxicity was prevented by increasing glyoxal metabolism with thiamine or NAD(P)H generators, and was increased in GSH- or thiamine-deficient hepatocytes. It was also found that feeding rats reduced thiamine levels in a diet high in simple sugars increased the number of aberrant crypt foci/colon in the absence of clinical evidence of beriberi. This was associated with decreased plasma thiamine and low erythrocyte transketolase activity. Western diets, which are frequently poor in thiamine and high in sugars, could result in increased levels of endogenous glyoxals, which in turn may lead to a predisposition to AGE (advanced glycation end-product)-related pathologies and neoplastic conditions.

Corpet and Pierre (D. E. Corpet and F. Pierre, Cancer Epidemiol. Biomark. Prev., 12: 391-400, 2003) have reviewed the prevention studies made with the azoxymethane rat and Min mouse colon cancer models, and have shown that many agents reduce the numbers of these experimental tumors. They suggest that agents with preventive activity with little or no toxicity should be evaluated in clinical intervention studies without delay. I think that the decision to proceed to a clinical trial is more complex, and involves an understanding of the safety of the agent and of the strength and consistency of the preclinical data. However, I am also impressed by the wide range of agents that have been found to affect the development of colon cancer in animals. This suggests that human colon cancer may be the consequence of many different dietary and lifestyle deficiencies, a view supported by the observation that normal mice develop colon cancer when fed diets deficient in several food components known to prevent tumors with the azoxymethane rat model [Newmark, H. L. et al., Carcinogenesis (Lond.), 22: 1871-1875, 2001]. There is a clear need to evaluate the preventive effects of additional combinations of these agents, identified perhaps from the Corpet and Pierre review (D. E. Corpet and F. Pierre, Cancer Epidemiol. Biomark. Prev., 12: 391-400, 2003), or from actual human high-risk diets. With diets that increase the risk of "spontaneous cancer" in hand, the stage would be set for assessing the most effective ways to reduce colon cancer risk, again first with animal studies, then clinical trials, and then perhaps population studies.

The similarity in lifestyle risk factors for the development of colorectal cancer (CRC) and type 2 diabetes suggests that there are common underlying pathogenic mechanisms. High-risk lifestyle factors may lead to insulin resistance that, through increased circulating levels of energy substrates, insulin, and insulin-like growth factor-1, may promote the development of CRC. The objective was to determine the extent to which direct and surrogate measures of insulin resistance correlate with multiplicity of aberrant crypt foci, which are putative precursors of CRC. Rats were initiated with the carcinogen azoxymethane, then fed low, intermediate, or high saturated fat diets. Metabolic parameters were assessed at 50 days and ACF at 100 days after initiation. Results indicate that CRC promotion was most strongly correlated with direct measures of insulin sensitivity as assessed with the hyperinsulinemic-euglycemic clamp (r = -0.52, P < 0.009). Practical surrogate measures of insulin resistance such as insulin levels at 180 min after an oral glucose load were strongly correlated with direct measures of insulin sensitivity (r = -0.61, P < 0.001) and with CRC promotion (r = 0.42, P = 0.044) in this animal model. Fasting levels of glucose, insulin, total insulin-like growth factor-1, nonesterified fatty acids, and triglyceride, as well as body weight and insulin sensitivity indices (such as fasting insulin resistance index, quantitative insulin sensitivity check index, homeostasis model assessment formula, insulin sensitivity index of glycemia, oral glucose insulin sensitivity, and composite insulin sensitivity index for the hepatic and peripheral tissues) were all less strongly correlated with direct measures of insulin sensitivity and all poorly correlated with CRC promotion in this animal model. These correlations do not prove causality, however, they suggest possible mechanisms linking diet, insulin resistance with its related parameters, and promotion of CRC.

It has recently been reported that 5% polyethylene glycol 8000 (PEG 8000; Mr 8000) in the diet markedly inhibits the development of colonic tumors in carcinogen-treated rats. To assess the possible use of this agent as a preventive or treatment agent for patients with familial adenomatous polyposis, we determined the effect of PEG 8000 on spontaneous carcinogenesis in the Min mouse. PEG at a 5% concentration in the diet of Min mice did not affect the number of small intestinal or cecal tumors but did increase the number of colon tumors and the number of animals with colonic tumors (2 of 18 versus 12 of 22 animals; P < 0.001). Although the chemopreventive effect of PEG 8000 in rats is remarkable, we suggest a cautious approach in long-term testing of PEG as a chemopreventive agent for subjects at risk for colonic neoplasia.

Two recent developments in cancer epidemiology and experimental carcinogenesis provide the basis for two possible mechanisms relating diet and colon cancer risk. The first development is the accumulating epidemiological evidence for an association between insulin resistance and colonic adenomas and cancers. This evidence suggests the following mechanism: the consumption of excess dietary energy results in the development of insulin resistance with increased circulating levels of insulin, triglycerides, and non-esterified fatty acids. These circulating factors subject colonic epithelial cells to a proliferative stimulus and also expose them to reactive oxygen intermediates. These long-term exposures result in the promotion of colon cancer. The second development is the continuing identification of agents that significantly inhibit experimental colon carcinogenesis. These observations suggest the following mechanism: focal loss of epithelial barrier function resulting from a failure of terminal differentiation results in the "leak" of a presently undefined toxin and a focal inflammatory response characterized by evidence of the activation of the COX-2 enzyme and an oxidative stress with the release of reactive oxygen intermediates. The resulting focal proliferation and mutagenesis give rise to aberrant crypt foci and adenomas. The process is inhibited by: (a) demulcents confined to the colonic lumen that "repair" the surface; (b) anti-inflammatory agents; or (c) antioxidants. The two mechanisms, i.e., insulin resistance acting throughout the body and focal epithelial barrier failure acting locally, can describe most of the known relationships between diet and colon cancer risk.

This work describes the application of high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy to the study of the thermal peroxidation of beef tallow and corn oil under standardized conditions. The approach provides a rapid, quantitative method for determining the degree of oxidation of unsaturated fatty acids in animal and vegetable fats and oils by quantitating the decreasing intensities of 1H-NMR peaks for allylic and olefinic protons in unsaturated fatty acid chains of triglycerides and the increasing peak intensities of hydroperoxide and saturated and alpha, beta-unsaturated aldehydic protons in relation to the less labile protons in the triglyceride molecule. Two-dimensional correlation spectroscopy analysis of highly oxidized beef tallow (180 degrees C for 24 h) suggested that the unsaturated aldehydes that persisted were apparently associated with carboxy groups.