Surgical incidents and their impact on operating theatre staff: qualitative study.

BACKGROUND: Surgical incidents can have significant effects on both patients and health professionals, including emotional distress and depression. The aim of this study was to explore the personal and professional impacts of surgical incidents on operating theatre staff. METHODS: Face-to-face semistructured interviews were conducted with a range of different healthcare professionals working in operating theatres, including surgeons and anaesthetists, operating department practitioners, and theatre nurses, and across different surgical specialties at five different hospitals. All interviews were audio recorded, transcribed verbatim, and analysed using an inductive thematic approach, which involved reading and re-reading the transcripts, assigning preliminary codes, and searching for patterns and themes within the codes, with the aid of NVivo 12 software. These emerging themes were discussed with the wider research team to gain their input. RESULTS: Some 45 interviews were conducted, generally lasting between 30 and 75 min. Three overarching themes emerged: personal and professional impact; impact of the investigation process; and positive consequences or impact. Participants recalled experiencing negative emotions following surgical incidents that depended on the severity of the incident, patient outcomes, and the support that staff received. A culture of blame, inadequate support, and lack of a clear and transparent investigative process appeared to worsen impact. CONCLUSION: The study indicated that more support is needed for operating theatre staff involved in surgical incidents. Greater transparency and better information during the investigation of such incidents for staff are still needed.

Digital technology to support lifestyle and health behaviour changes in surgical patients: systematic review.

BACKGROUND: Digital technologies (such as smartphone applications, activity trackers, and e-learning platforms) have supported patients with long-term conditions to change their lifestyle health behaviours. The aim of this study was to examine the effectiveness of digital technologies in supporting patients undergoing elective surgery to change their health behaviours. METHODS: A systematic review was conducted of articles reporting a digital intervention supporting behaviour change in adult patients who underwent elective bariatric, oncological or orthopaedic surgery. MEDLINE, Embase, CINAHL, PsycINFO, Web of Science, and Scopus were searched from inception to March 2019 for quantitative intervention studies with a specific focus on physical activity, dietary intake, and weight loss in patients before and after surgery (PROSPERO: CRD42019127972). The Joanna Briggs Institute critical appraisal checklist was used to assess study quality. RESULTS: Of 3021 citations screened, 17 studies were included comprising 4923 surgical patients; these included experimental (pre-post design, feasibility studies, and RCTs) and observational studies. Three factors were identified as effective for supporting health behaviour change in elective surgical populations: digital technology delivery, implementation, and theoretical underpinning. Six of eight studies that referred to behaviour change theories observed significant improvements in health behaviour relating to reduced weight regain, and improved lifestyle choices for physical activity and diet. Meta-analysis was not possible because of heterogeneous outcome measures. CONCLUSION: Digital technologies may effectively support behavioural change in patients undergoing elective surgery.

Systematic review of psychological, emotional and behavioural impacts of surgical incidents on operating theatre staff.

BACKGROUND: Adverse surgical incidents affect both patients and health professionals. This study sought to explore the effect of surgical incidents on operating theatre staff and their subsequent behaviours. METHODS: Eligible studies were primary research or reviews that focused on the effect of incidents on operating theatre staff in primary, secondary or tertiary care settings. MEDLINE, Embase, CINALH and PsycINFO were searched. A data extraction form was used to capture pertinent information from included studies and the Critical Appraisal Skills Programme (CASP) tool to appraise their quality. PRISMA-P reporting guidelines were followed and the review is registered with PROSPERO. RESULTS: A total of 3918 articles were identified, with 667 duplicates removed and 3230 excluded at the title, abstract and full-text stages. Of 21 included articles, eight focused on the impact of surgical incidents on surgeons and anaesthetists. Only two involved theatre nurses and theatre technicians. Five key themes emerged: the emotional impact on health professionals, organization culture and support, individual coping strategies, learning from surgical complications and recommended changes to practice. CONCLUSION: Health professionals suffered emotional distress and often changed their behaviour following a surgical incident. Both organizations and individual clinicians can do a great deal to support staff in the aftermath of serious incidents.

Drug allergies documented in electronic health records of a large healthcare system.

BACKGROUND: The prevalence of drug allergies documented in electronic health records (EHRs) of large patient populations is understudied. OBJECTIVE: We aimed to describe the prevalence of common drug allergies and patient characteristics documented in EHRs of a large healthcare network over the last two decades. METHODS: Drug allergy data were obtained from EHRs of patients who visited two large tertiary care hospitals in Boston from 1990 to 2013. The prevalence of each drug and drug class was calculated and compared by sex and race/ethnicity. The number of allergies per patient was calculated and the frequency of patients having 1, 2, 3…, or 10+ drug allergies was reported. We also conducted a trend analysis by comparing the proportion of each allergy to the total number of drug allergies over time. RESULTS: Among 1 766 328 patients, 35.5% of patients had at least one reported drug allergy with an average of 1.95 drug allergies per patient. The most commonly reported drug allergies in this population were to penicillins (12.8%), sulfonamide antibiotics (7.4%), opiates (6.8%), and nonsteroidal anti-inflammatory drugs (NSAIDs) (3.5%). The relative proportion of allergies to angiotensin-converting enzyme (ACE) inhibitors and HMG CoA reductase inhibitors (statins) have more than doubled since early 2000s. Drug allergies were most prevalent among females and white patients except for NSAIDs, ACE inhibitors, and thiazide diuretics, which were more prevalent in black patients. CONCLUSION: Females and white patients may be more likely to experience a reaction from common medications. An increase in reported allergies to ACE inhibitors and statins is noteworthy.

Standard Information Models for Representing Adverse Sensitivity Information in Clinical Documents.

BACKGROUND: Adverse sensitivity (e.g., allergy and intolerance) information is a critical component of any electronic health record system. While several standards exist for structured entry of adverse sensitivity information, many clinicians record this data as free text. OBJECTIVES: This study aimed to 1) identify and compare the existing common adverse sensitivity information models, and 2) to evaluate the coverage of the adverse sensitivity information models for representing allergy information on a subset of inpatient and outpatient adverse sensitivity clinical notes. METHODS: We compared four common adverse sensitivity information models: Health Level 7 Allergy and Intolerance Domain Analysis Model, HL7-DAM; the Fast Healthcare Interoperability Resources, FHIR; the Consolidated Continuity of Care Document, C-CDA; and OpenEHR, and evaluated their coverage on a corpus of inpatient and outpatient notes (n = 120). RESULTS: We found that allergy specialists' notes had the highest frequency of adverse sensitivity attributes per note, whereas emergency department notes had the fewest attributes. Overall, the models had many similarities in the central attributes which covered between 75% and 95% of adverse sensitivity information contained within the notes. However, representations of some attributes (especially the value-sets) were not well aligned between the models, which is likely to present an obstacle for achieving data interoperability. Also, adverse sensitivity exceptions were not well represented among the information models. CONCLUSIONS: Although we found that common adverse sensitivity models cover a significant portion of relevant information in the clinical notes, our results highlight areas needed to be reconciled between the standards for data interoperability.

Immune requirements for protective Th17 recall responses to Mycobacterium tuberculosis challenge.

Tuberculosis (TB) vaccine development has focused largely on targeting T helper type 1 (Th1) cells. However, despite inducing Th1 cells, the recombinant TB vaccine MVA85A failed to enhance protection against TB disease in humans. In recent years, Th17 cells have emerged as key players in vaccine-induced protection against TB. However, the exact cytokine and immune requirements that enable Th17-induced recall protection remain unclear. In this study, we have investigated the requirements for Th17 cell-induced recall protection against Mycobacterium tuberculosis (Mtb) challenge by utilizing a tractable adoptive transfer model in mice. We demonstrate that adoptive transfer of Mtb-specific Th17 cells into naive hosts, and upon Mtb challenge, results in Th17 recall responses that confer protection at levels similar to vaccination strategies. Importantly, although interleukin (IL)-23 is critical, IL-12 and IL-21 are dispensable for protective Th17 recall responses. Unexpectedly, we demonstrate that interferon-γ (IFN-γ) produced by adoptively transferred Th17 cells impairs long-lasting protective recall immunity against Mtb challenge. In contrast, CXCR5 expression is crucial for localization of Th17 cells near macrophages within well-formed B-cell follicles to mediate Mtb control. Thus, our data identify new immune characteristics that can be harnessed to improve Th17 recall responses for enhancing vaccine design against TB.

Discussion of "Attitude of physicians towards automatic alerting in computerized physician order entry systems".

With these comments on the paper "Attitude of Physicians Towards Automatic Alerting in Computerized Physician Order Entry Systems", written by Martin Jung and co-authors, with Dr. Elske Ammenwerth as senior author [1], the journal wants to stimulate a broad discussion on computerized physician order entry systems. An international group of experts have been invited by the editor of Methods to comment on this paper. Each of the invited commentaries forms one section of this paper.

Interleukin-17-dependent CXCL13 mediates mucosal vaccine-induced immunity against tuberculosis.

The variable efficacy of tuberculosis (TB) vaccines and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) emphasize the urgency for not only generating new and more effective vaccines against TB but also understanding the underlying mechanisms that mediate vaccine-induced protection. We demonstrate that mucosal adjuvants, such as type II heat labile enterotoxin (LT-IIb), delivered through the mucosal route induce pulmonary Mtb-specific T helper type 17 (Th17) responses and provide vaccine-induced protection against Mtb infection. Importantly, protection is interferon-γ (IFNγ)-independent but interleukin-17 (IL-17)-dependent. Our data show that IL-17 mediates C-X-C motif chemokine ligand 13 (CXCL13) induction in the lung for strategic localization of proinflammatory cytokine-producing CXCR5+ (C-X-C motif chemokine receptor 5-positive) T cells within lymphoid structures, thereby promoting early and efficient macrophage activation and the control of Mtb. Our studies highlight the potential value of targeting the IL-17-CXCL13 pathway rather than the IFNγ pathway as a new strategy to improve mucosal vaccines against TB.

Tissue-specific dysregulation of cortisol metabolism in equine laminitis.

REASONS FOR PERFORMING STUDY: The role of glucocorticoids (GCs) in the pathogenesis of laminitis is incompletely understood. Local tissue activity of GC is regulated by the steroid converting enzyme, 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1). Changes in integumentary (skin and hoof lamellar) 11beta-HSD activity occurring during laminitis could affect the extent to which GCs are involved in its development. HYPOTHESIS: That changes in integumentary 11beta-HSD-1 activity associated with the laminitic condition would lead to elevated local tissue levels of GCs, which could subsequently contribute, through paracrine and autocrine mechanisms, to the further development of laminitis; and that similar changes in 11beta-HSD-1 activity would be evident in both skin and hoof lamellar tissue. METHODS: Activity of 11beta-HSD-1 was determined in skin and hoof lamellar tissue specimens obtained from normal and laminitic horses using a radiometric assay. Skin samples were obtained from 10 normal horses and from 10 horses before and after induction of acute laminitis following administration of starch via nasogastric tube. Hoof lamellar samples were obtained from 10 normal horses, 10 horses following induction of acute laminitis and 4 chronically-foundered horses. Bidirectional 11beta-HSD-1 activity was measured in both skin and lamellar tissues. RESULTS: 11-ketoreductase activity exceeded 11beta-dehydrogenase activity in both skin and lamellar tissues. Cutaneous activity was higher than lamellar 11beta-HSD-1 activity in all groups. Both ketoreductase and dehydrogenase activity increased in skin and lamellae following experimental induction of acute laminitis, but the increase in ketoreductase activity was substantially greater than that for dehydrogenase in the lamellae. Induction of acute laminitis was attended by increases of 227 and 220% in cutaneous dehydrogenase and ketoreductase activity, respectively, and 173 and 398% in lamellar dehydrogenase and ketoreductase activity, respectively (P<0.05). CONCLUSIONS: The 11-ketoreductase moiety of 11beta-HSD-1 plays a role in equine skin and hoof lamellae regarding the regulation of local glucocorticoid activity. Increased 11-ketoreductase activity will lead to increased local tissue GC activity by virtue of conversion of cortisone to cortisol. POTENTIAL RELEVANCE: The laminitic condition is attended by integumentary biochemical changes that enhance the local concentration of cortisol, especially in the hoof lamellar interface. Through multiple and diverse actions, increased local GC activity contributes to the pathogenesis and morbidity associated with laminitis. Pharmacological manipulation of 11beta-HSD-1 deserves further investigation regarding the prevention and treatment of laminitis.

Extra-adrenal mineralocorticoids and cardiovascular tissue.

In experimental models where chronic inappropriate (relative to sodium intake and intravascular volume) elevations in circulating mineralocorticoids (aldosterone or deoxycorticosterone) are created, a reactive fibrosis with vascular remodeling is observed in systemic organs and the heart. Until recently, it was assumed that aldosterone was derived solely from adrenal glands via the circulation; however, there is now convincing evidence that cells of the heart and vasculature express genes responsible for the formation of both aldosterone and corticosterone and are capable of producing these steroids. Vascular endothelial and smooth muscle cells express CYP11B1 and CYP11B2, genes responsible for 11 beta -hydroxylase and aldosterone synthase, respectively. Furthermore, smooth muscle cells elaborate aldosterone. There is evidence that similar regulatory mechanisms operate in vascular cells as in adrenal cortex, since aldosterone synthase and 11 beta -hydroxylase expression are differentially modulated by low sodium/high potassium, angiotensin II and ACTH. It is likely that such localized corticosteroid production also occurs at sites of tissue repair, where populations of collagen-producing myofibroblasts, nourished by a neovasculature, predominate. Using a subcutaneous pouch model of granulation tissue we have obtained compelling data which would support such a notion. The mineralocorticoid receptor antagonist, spironolactone, severely attenuates pouch formation over a 2-week period and significantly reduces pouch wall hydroxyproline concentration. This effect is apparent even following adrenalectomy, when circulating corticosteroids are undetectable; however, with adrenalectomy alone, pouch formation is barely affected. This we took to be a possible indication of an effect of local, non-adrenal steroids in maintaining pouch tissue. Spironolactone inhibits angiogenesis. A recent clinical study demonstrates the efficacy of low-dose spironolactone in enhancing survival in patients with advanced chronic cardiac failure. Although it is not known how spironolactone brings about such an improvement in survival, we would propose that inhibition of fibrous tissue formation and/or angiogenesis might be important contributory factors. Further studies are required to address the relative contributions of circulating vs local aldosterone in promoting normal vs pathologic connective tissue formation.

Inhibition of tissue repair by spironolactone: role of mineralocorticoids in fibrous tissue formation.

Mineralocorticoids have been implicated in promoting fibrous tissue formation in various organs. In the present study, we sought to address the potential contribution of mineralocorticoids to fibrous tissue formation using a skin pouch model which has proved valuable for the analysis of inflammatory and wound healing responses. Skin pouches were induced in rats by administration of a phorbol ester, croton oil (0.5 ml of a 1% solution). After 2 weeks, rats were killed and intact pouch tissue collected. Pouch weights of control and aldosterone-treated (0.75 microg/h via osmotic minipump) rats were similar (3.33 +/- 0.44 g vs. 3.70 +/- 0.28 g respectively). However, pouch weights were reduced by more than 50% in spironolactone-treated (25 mg/day powdered in food) animals (1.62 +/- 0.22 g and 1.27 +/- 0.23 g respectively in aldosterone and spironolactone alone groups). To ascertain the effects of different treatments on collagen accumulation, hydroxyproline concentration was measured. Compared with controls, hydroxyproline concentration was significantly reduced following spironolactone treatment (17.1 +/- 0.08 vs. 7.5 +/- 2.0 microg/mg dry wt, respectively, p < 0.01). This response to spironolactone was negated by coadministration of aldosterone (hydroxyproline concentration was 18.6 +/- 2.1 microg/mg dry wt). Following bilateral adrenalectomy, spironolactone reduced pouch weight and hydroxyproline concentration, which was not the case for adrenalectomy alone. Two week aldosterone administration in uninephrectomized rats on high salt diet was deemed ineffective in modulating pouch development (pouch wet wts were 3.48 +/- 0.4 g vs. 3.00 +/- 0.19 g in controls and aldosterone-treated rats, respectively). Mineralocorticoid receptor expression in pouch tissue was demonstrated by RT/PCR. Furthermore, NADP+-dependent 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity was detected in pouch tissue, together with lower levels of NAD+-dependent 11beta-HSD2. Spironolactone (p < 0.05) significantly reduced 11beta-HSD1 activity compared with controls. Thus, fibrous tissue possesses requisite components of MC action, and antagonism of mineralocorticoid receptors by spironolactone attenuates its formation. Pouch formation is under the influence of circulating MC and, we would like to propose, is also mediated through corticosteroids generated de novo at the site of tissue repair.

High affinity NAD(+)-dependent 11 beta-hydroxysteroid dehydrogenase in the human heart.

Receptor-ligand binding is an essential component of mineralocorticoid (MC) activity in target tissues. Detection of type 1 mineralocorticoid receptors (MR) in cardiac tissue is therefore suggestive that, like kidney, the heart is MC responsive. The presence of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) within MC responsive tissue is essential to prevent saturation of MR by glucocorticoids. Using both high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC), we have found that a high-affinity species of 11 beta-HSD predominates within human heart. Although two 11 beta-HSD isoforms were detected in human cardiac tissues, the activity of high-affinity (type 2) 11 beta-HSD was found to be at least twice that of low affinity (type 1) 11 beta-HSD. Human cardiac type 2 11 beta-HSD possesses characteristics identical to the high-affinity enzyme of distal renal tubules; 11 beta-dehydrogenation of corticosterone or cortisol to their 11-keto metabolites is NAD(+)-dependent and, with corticosterone as substrate, the enzyme has a nanomolar Km (15.1 nM as determined by Lineweaver-Burke analysis). Furthermore, its activity is unidirectional; corticosterone and cortisol are 11 beta-dehydrogenated to inactive 11-keto metabolites, whereas 11-oxoreductase activity (conversion of 11-dehydrocorticosterone and cortisone to corticosterone and cortisol, respectively) is absent. RT/PCR analysis, using primers complementary to the human renal type 2 11 beta-HSD sequence, demonstrated that the high-affinity species of 11 beta-HSD expressed in human heart is indeed the same enzyme as that produced in the kidney. These findings strongly suggest that, as is the case in the distal portion of the nephron, type 2 11 beta-HSD plays an important role in the human heart to promote glucocorticoid metabolism and to confer MC specificity upon MR.

Vascular remodeling and mineralocorticoids.

Circulating mineralocorticoid hormones are so named because of their important homeostatic properties that regulate salt and water balance via their action on epithelial cells. A broader range of functions in nonclassic target cellular sites has been proposed for these steroids and includes their contribution to wound healing following injury. A chronic, inappropriate (relative to intravascular volume and dietary sodium intake) elevation of these circulating hormones evokes a wound healing response in the absence of tissue injury--a wound healing response gone awry. The adverse remodeling of vascularized tissues seen in association with chronic mineralocorticoid excess is the focus of this review.

Chronic mineralocorticoid excess and cardiovascular remodeling.

Chronic mineralocorticoid (MC) excess, whether due to elevated plasma aldosterone (ALDO) or deoxycorticosterone (DOC), is associated with a perivascular fibrosis of systemic and coronary arterioles. This remodeling of resistance vessels contributes to the appearance of hypertension. Chronic MC excess is also accompanied by cardiac myocyte necrosis, secondary to myocardial potassium depletion, and a subsequent reparative fibrosis that appears in the normotensive, nonhypertrophied right and hypertensive, hypertrophied left ventricles. Fibrosis contributes to the appearance of ventricular arrhythmias and dysfunction. Herein, clinical and experimental evidence linking chronic, inappropriate (relative to dietary sodium) elevations in circulating ALDO and DOC with these reactive and reparative forms of fibrous tissue formation in the heart and other tissues is presented.

Species diversity of 11 beta-hydroxysteroid dehydrogenase in the cardiovascular system.

Mineralocorticoid (MC) receptors are found in classic (e.g., kidney) and nonclassic (e.g., heart and aorta) tissues. MC receptor specificity at either site is conferred by the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which metabolizes glucocorticoids to inactive 11-ketosteroids. Because the heart and vasculature may be target tissues for aldosterone, this study was undertaken. Its objectives were to measure 11 beta-HSD at these sites and to compare levels of activity in the atria and ventricles of different species. Toward this end we first determined levels of plasma corticosterone (B) and cortisol (F) (by using radioimmunoassay), in a variety of species, for subsequent correlation with cardiac 11 beta-HSD activity. 11 beta-Dehydrogenation of glucocorticoids was then assayed in ventricles and atria as well as aorta. Tissue homogenates containing 1 to 5 mg protein were incubated for 1 hour in the presence of 1 muCi 5 x 10(-9) mol/L tritiated B or tritiated F and 5 x 10(-4) mol/L oxidized nicotinamide adenine dinucleotide phosphate. Steroid separation and quantitation were achieved by using reverse-phase high-performance liquid chromatography coupled to an online radioisotope detector. For species in which B circulates at relatively high concentrations (rat, rabbit, pig), high levels of dehydrogenation of B to 11-dehydrocorticosterone (A) were observed in both atria and ventricles. Overall, cardiac B to A conversion levels corresponded to between 0.3 and 0.4 pmol A formed/mg protein/hr. 11 beta-HSD activity was also detected in the aorta.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid metabolism in the cardiac interstitium: 11 beta-hydroxysteroid dehydrogenase activity in cardiac fibroblasts.

The interstitial fibrosis seen in the heart and systemic organs in states of primary or secondary mineralocorticoid excess suggests that fibroblasts are responsive to mineralocorticoid. In vitro studies demonstrating increased fibroblast collagen synthesis in response to MC are consonant with this view. The nicotinamide-adenine dinucleotide phosphate(+)-dependent enzyme 11 beta-hydroxysteroid dehydrogenase converts the glucocorticoids corticosterone and cortisol to the inactive metabolites 11-dehydrocorticosterone and cortisone, respectively, conferring mineralocorticoid specificity to the cells within which it is active. We investigated the presence of 11 beta-hydroxysteroid dehydrogenase in sonicates of cultured vascular endothelial cells and cardiac fibroblasts by incubating sonicates for 1 hour in the presence of 5 x 10(-9) mol/L tritiated corticosterone or tritiated cortisol (1 microCi) and using reverse-phase high-performance liquid chromatography coupled to an on-line radioisotope detector for steroid separation and quantitation. Extracts of bovine endothelial cells showed no enzymatic activity with either substrate, whereas extracts of rat cardiac fibroblasts readily converted corticosterone to 11-dehydrocorticosterone, even in the absence of exogenous nicotinamide-adenine dinucleotide phosphate+ (10% conversion). When 5 x 10(-4) mol/L nicotinamide-adenine dinucleotide phosphate+ was added to sonicated fibroblasts, conversion increased to 50%, corresponding to 12 pmol 11-dehydrocorticosterone formed/mg protein. Conversion of cortisol to cortisone was not observed in fibroblast or endothelial cell extracts. Significant levels of corticosterone to 11-dehydrocorticosterone conversion (0.14 pmol/10(6) cells/hour) were detected in intact fibroblasts, but no 11-dehydrogenation of corticosterone was observed in intact endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

Glycation of lens proteins by the oxidation products of ascorbic acid.

Bovine lens water-soluble proteins were incubated with [I-14C]ascorbic acid (ASA) for 6 days, and the incorporation into protein was measured at daily intervals. Aliquots were also withdrawn to determine the distribution of label among the various ASA oxidation products. A linear incorporation into protein was observed in the presence of NaCNBH3, however, little or no incorporation was seen in its absence. TLC analysis showed a complete loss of ASA by day 3, whereas both dehydroascorbate (DHA) and diketogulonic acid (DKG) remained constant for 6 days, consistent with the linear incorporation into protein. The amino acid composition of the proteins glycated in the presence of NaCNBH3 was identical to controls except for a 70% reduction in lysine residues and a corresponding increase in an unknown product which eluted slightly earlier than methionine. In the absence of NaCNBH3 lysine decreased linearly to 20% with an additional decrease in arginine and histidine at later times concurrent with protein crosslinking. DHA and DKG were prepared and incubated directly with lens proteins for an 8 day period. Both compounds glycated lens protein as evidenced by an increased binding to a boronate affinity column. SDS-PAGE showed that both compounds were also capable of causing protein crosslinking. DHA is apparently capable of reacting directly with protein since glycation was observed with the ASA analog, reductic acid, which can be oxidized to dehydroreductic acid, but which cannot be hydrolyzed to an open chain structure. DHA also produced a lysine adduct which was not obtained with DKG, supporting the idea that both species have glycating ability.