Mortality attributable to diets low in fruits, vegetables, and whole grains in Brazil in 2019: evidencing regional health inequalities.

OBJECTIVE: The objective of this study was to describe non-communicable disease (NCD) mortality attributable to diets low in whole grains, fruits, and vegetables in Brazil in 2019. STUDY DESIGN: Ecological study. METHODS: Data from the Global Burden of Disease 2019 for adults aged ≥25 years of both sexes in Brazil and its 27 states were used to estimate the intake of fruits, vegetables, and whole grains; the NCD mortality attributable to these dietary risk factors; and the correlation between socio-demographic index (SDI), the age-standardised mortality rate (ASMR) per 100,000 population, and intake. RESULTS: The Brazilian population had suboptimal consumption of fruits, vegetables, and whole grains, and 62,439 NCD deaths were attributable to these three dietary risk factors in 2019. The highest ASMRs were found for diets low in whole grains (14.4, 95% uncertainty interval [95% UI]: 7.8-18.4), followed by diets low in vegetables (7.6, 95% UI: 4.8-10.3) and fruits (5.0, 95% UI: 3.2-7.0). A similar ranking was observed for all Brazilian states. The SDI was negatively correlated with ASMRs and was positively correlated with the investigated dietary risks. The population from the Northeast and North states presented the lowest SDI and the highest NCD ASMRs attributable to diets low in fruits, vegetables, and whole grains and consumed less of all three health foods. CONCLUSION: Diets low in fruits, vegetables, and mainly whole grains substantially contributed to NCD mortality in Brazil, especially in states with low SDI. Our findings support the need to target food interventions to reduce regional health inequalities within the country.

Coronary heart disease mortality is decreasing in Argentina, and Colombia, but keeps increasing in Mexico: a time trend study.

BACKGROUND: Mortality rates due to coronary heart disease (CHD) have decreased in most countries, but increased in low and middle-income countries. Few studies have analyzed the trends of coronary heart disease mortality in Latin America, specifically the trends in young-adults and the effect of correcting these comparisons for nonspecific causes of death (garbage codes). The objective of this study was to describe and compare standardized, age-specific, and garbage-code corrected mortality trends for coronary heart disease from 1985 to 2015 in Argentina, Colombia, and Mexico. METHODS: Deaths from coronary heart disease were grouped by country, year of registration, sex, and 10-year age bands to calculate age-adjusted and age and sex-specific rates for adults aged ≥25. We corrected for garbage-codes using the methodology proposed by the Global Burden of Disease. Finally, we fitted Joinpoint regression models. RESULTS: In 1985, age-standardized mortality rates per 100,000 population were 136.6 in Argentina, 160.6 in Colombia, and 87.51 in Mexico; by 2015 rates decreased 51% in Argentina and 6.5% in Colombia, yet increased by 61% in Mexico, where an upward trend in mortality was observed in young adults. Garbage-code corrections produced increases in mortality rates, particularly in Argentina with approximately 80 additional deaths per 100,000, 14 in Colombia and 13 in Mexico. CONCLUSIONS: Latin American countries are at different stages of the cardiovascular disease epidemic. Garbage code correction produce large changes in the mortality rates in Argentina, yet smaller in Mexico and Colombia, suggesting garbage code corrections may be needed for specific countries. While coronary heart disease (CHD) mortality is falling in Argentina, modest falls in Colombia and substantial increases in Mexico highlight the need for the region to propose and implement population-wide prevention policies.

Adverse risk factor trends limit gains in coronary heart disease mortality in Barbados: 1990-2012.

BACKGROUND: Although most countries face increasing population levels of obesity and diabetes their effect on coronary heart disease (CHD) mortality has not been often studied in small island developing states (SIDs) where obesity rates are among the highest in the world. We estimated the relative contributions of treatments and cardiovascular risk factors to the decline in CHD mortality from 1990 to 2012 in the Caribbean island, Barbados. METHODS: We used the IMPACT CHD mortality model to estimate the effect of increased coverage of effective medical/surgical treatments and changes in major CHD risk factors on mortality trends in 2012 compared with 1990. We calculated deaths prevented or postponed (DPPs) for each model risk factor and treatment group. We obtained data from WHO Mortality database, population denominators from the Barbados Statistical Service stratified by 10-year age group (ages 25-34 up to 85 plus), population-based risk factor surveys, Global Burden of Disease and Barbados' national myocardial infarction registry. Monte Carlo probabilistic sensitivity analysis was performed. RESULTS: In 1990 the age-standardized CHD mortality rate was 109.5 per 100,000 falling to 55.3 in 2012. Implementation of effective treatment accounted for 56% DPPs (95% (Uncertainty Interval (UI) 46%, 68%), mostly due to the introduction of treatments immediately after acute myocardial infarction (AMI) (14%) and unstable angina (14%). Overall, risk factors contributed 19% DPPs (95% UI 6% to 34%) mostly attributed to decline in cholesterol (18% DPPs, 95% UI 12%, 26%). Adverse trends in diabetes: 14% additional deaths(ADs) 95% UI 8% to 21% ADs) and BMI (2% ADs 95%UI 0 to 5% ADs) limited potential for risk factor gains. CONCLUSIONS: Given the significant negative impact of obesity/diabetes on mortality in this analysis, research that explores factors affecting implementation of evidenced-based preventive strategies is needed. The fact that most of the decline in CHD mortality in Barbados was due to treatment provides an example for SIDs about the advantages of universal access to care and treatment.

Assessment of cardiovascular risk in Tunisia: applying the Framingham risk score to national survey data.

OBJECTIVE: This paper aims to assess the socioeconomic determinants of a high 10 year cardiovascular risk in Tunisia. SETTING: We used a national population based cross sectional survey conducted in 2005 in Tunisia comprising 7780 subjects. We applied the non-laboratory version of the Framingham equation to estimate the 10 year cardiovascular risk. PARTICIPANTS: 8007 participants, aged 35-74 years, were included in the sample but effective exclusion of individuals with cardiovascular diseases and cancer resulted in 7780 subjects (3326 men and 4454 women) included in the analysis. RESULTS: Mean age was 48.7 years. Women accounted for 50.5% of participants. According to the Framingham equation, 18.1% (17.25-18.9%) of the study population had a high risk (≥20% within 10 years). The gender difference was striking and statistically significant: 27.2% (25.7-28.7%) of men had a high risk, threefold higher than women (9.7%; 8.8-10.5%). A higher 10 year global cardiovascular risk was associated with social disadvantage in men and women; thus illiterate and divorced individuals, and adults without a professional activity had a significantly higher risk of developing a cardiovascular event in 10 years. Illiterate men were at higher risk than those with secondary and higher education (OR=7.01; 5.49 to 9.14). The risk in illiterate women was more elevated (OR=13.57; 7.58 to 24.31). Those living in an urban area had a higher risk (OR=1.45 (1.19 to 1.76) in men and OR=1.71 (1.35 to 2.18) in women). CONCLUSIONS: The 10 year global cardiovascular risk in the Tunisian population is already substantially high, affecting almost a third of men and 1 in 10 women, and concentrated in those more socially disadvantaged.

Exploring potential mortality reductions in 9 European countries by improving diet and lifestyle: A modelling approach.

BACKGROUND: Coronary heart disease (CHD) death rates have fallen across most of Europe in recent decades. However, substantial risk factor reductions have not been achieved across all Europe. Our aim was to quantify the potential impact of future policy scenarios on diet and lifestyle on CHD mortality in 9 European countries. METHODS: We updated the previously validated IMPACT CHD models in 9 European countries and extended them to 2010-11 (the baseline year) to predict reductions in CHD mortality to 2020(ages 25-74years). We compared three scenarios: conservative, intermediate and optimistic on smoking prevalence (absolute decreases of 5%, 10% and 15%); saturated fat intake (1%, 2% and 3% absolute decreases in % energy intake, replaced by unsaturated fats); salt (relative decreases of 10%, 20% and 30%), and physical inactivity (absolute decreases of 5%, 10% and 15%). Probabilistic sensitivity analyses were conducted. RESULTS: Under the conservative, intermediate and optimistic scenarios, we estimated 10.8% (95% CI: 7.3-14.0), 20.7% (95% CI: 15.6-25.2) and 29.1% (95% CI: 22.6-35.0) fewer CHD deaths in 2020. For the optimistic scenario, 15% absolute reductions in smoking could decrease CHD deaths by 8.9%-11.6%, Salt intake relative reductions of 30% by approximately 5.9-8.9%; 3% reductions in saturated fat intake by 6.3-7.5%, and 15% absolute increases in physical activity by 3.7-5.3%. CONCLUSIONS: Modest and feasible policy-based reductions in cardiovascular risk factors (already been achieved in some other countries) could translate into substantial reductions in future CHD deaths across Europe. However, this would require the European Union to more effectively implement powerful evidence-based prevention policies.

Explaining the trends in coronary heart disease mortality in Barbados: 1990–2012

OBJECTIVE: To describe the relative contributions of medical treatments and major cardiovascular risk factors to the decline in coronary heart disease (CHD) mortality from1990 to 2012 in Barbados. SUBJECTS AND METHODS: We used the IMPACT CHD mortality model to estimate the effect of improvement in uptake or efficacy of medical/surgical treatments, versus changes in major CHD risk factors on mortality trends. We obtained death data from the World Health Organization(WHO) mortality database and population denominators, stratified by age and gender from the Barbados Statistical Service. Cardiovascular risk factors and treatment data were obtained from published studies, population-based risk factor surveys, Barbados’ national myocardial infarction registry and retrospective chart reviews. RESULTS: In 1990, the age-standardized CHD mortality rate was 109.5 per 100 000, falling to 55.3 in 2012, representing a 46.1% decline in CHD deaths. This resulted in139 fewer deaths observed in 2012 versus the number expected had the rate remained as in 1990. The model indicated that 61% (n = 84) of these deaths were prevented or postponed (DPPs) because of implementation of treatment. Changes in risk factors accounted for 14% of the overall decline (19 DPPs). Improvements in cholesterol, physical inactivity, smoking and fruit/vegetable intake accounted for 51 DPPs; worsening systolic bloodpressure, diabetes and obesity levels were responsible for 32 additional deaths in 2012. CONCLUSIONS: Treatments accounted for approximately two-thirds of the mortality reduction. More effective prevention policies are urgently needed.

The contribution of primary prevention medication and dietary change in coronary mortality reduction in England between 2000 and 2007: a modelling study.

OBJECTIVE: To analyse the falls in coronary heart disease (CHD) mortality in England between 2000 and 2007 and quantify the relative contributions from preventive medications and population-wide changes in blood pressure (BP) and cholesterol levels, particularly by exploring socioeconomic inequalities. DESIGN: A modelling study. SETTING: Sources of data included controlled trials and meta-analyses, national surveys and official statistics. PARTICIPANTS: English population aged 25+ in 2000-2007. MAIN OUTCOME MEASURES: Number of deaths prevented or postponed (DPPs) in 2007 by socioeconomic status. We used the IMPACTSEC model which applies the relative risk reduction quantified in previous randomised controlled trials and meta-analyses to partition the mortality reduction among specific treatments and risk factor changes. RESULTS: Between 2000 and 2007, approximately 20 400 DPPs were attributable to reductions in BP and cholesterol in the English population. The substantial decline in BP was responsible for approximately 13 000 DPPs. Approximately 1800 DPPs came from medications and some 11 200 DPPs from population-wide changes. Reduction in population BP prevented almost twofold more deaths in the most deprived quintile compared with the most affluent. Reduction in cholesterol resulted in approximately 7400 DPPs; approximately 5300 DPPs were attributable to statin use and approximately 2100 DPPs to population-wide changes. Statins prevented almost 50% more deaths in the most affluent quintile compared with the most deprived. Conversely, population-wide changes in cholesterol prevented threefold more deaths in the most deprived quintile compared with the most affluent. CONCLUSIONS: Population-wide secular changes in systolic blood pressure (SBP) and cholesterol levels helped to substantially reduce CHD mortality and the associated socioeconomic disparities. Mortality reductions were, in absolute terms, greatest in the most deprived quintiles, mainly reflecting their bigger initial burden of disease. Statins for high-risk individuals also made an important contribution but maintained socioeconomic inequalities. Our results strengthen the case for greater emphasis on preventive approaches, particularly population-based policies to reduce SBP and cholesterol.

Trends and future projections of the prevalence of adult obesity in Saudi Arabia, 1992-2022.

The prevalence of obesity among adults in Saudi Arabia increased from 22% in 1990-1993 to 36% in 2005, and future projections of the prevalence of adult obesity are needed by health policy-makers. In a secondary analysis of published data, a number of assumptions were applied to estimate the trends and projections in the age-and sex-specific prevalence of adult obesity in Saudi Arabia over the period 1992-2022. Five studies conducted between 1989 and 2005 were eligible for inclusion, using body mass index (BMI) ≥ 30 kg/m(2) to define obesity. The overall prevalence of obesity was projected to increase from around 12% in 1992 to 41% by 2022 in men, and from 21% to 78% in women. Women had much higher projected prevalence than men, particularly in the age groups 35-44, 45-54 and 55-64 years. Effective national strategies are needed to reduce or halt the projected rise in obesity prevalence.

IMPACT: a generic tool for modelling and simulating public health policy.

BACKGROUND: Populations are under-served by local health policies and management of resources. This partly reflects a lack of realistically complex models to enable appraisal of a wide range of potential options. Rising computing power coupled with advances in machine learning and healthcare information now enables such models to be constructed and executed. However, such models are not generally accessible to public health practitioners who often lack the requisite technical knowledge or skills. OBJECTIVES: To design and develop a system for creating, executing and analysing the results of simulated public health and healthcare policy interventions, in ways that are accessible and usable by modellers and policy-makers. METHODS: The system requirements were captured and analysed in parallel with the statistical method development for the simulation engine. From the resulting software requirement specification the system architecture was designed, implemented and tested. A model for Coronary Heart Disease (CHD) was created and validated against empirical data. RESULTS: The system was successfully used to create and validate the CHD model. The initial validation results show concordance between the simulation results and the empirical data. CONCLUSIONS: We have demonstrated the ability to connect health policy-modellers and policy-makers in a unified system, thereby making population health models easier to share, maintain, reuse and deploy.

Coronary heart disease mortality trends in the Netherlands 1972-2007.

BACKGROUND: Coronary heart disease (CHD) mortality has steadily declined since the early 1970s in the Netherlands. However, in some Western countries the rate of decline in younger groups may be starting to plateau or even rise. OBJECTIVE: To examine trends in age-specific CHD mortality rates among Dutch adults from 1972 to 2007, with a particular focus on recent trends for the younger age groups METHODS: Data for all CHD deaths (1972-2007) in the Netherlands were grouped by year, sex, age. A joinpoint regression was fitted to each age-sex-group to detect points in time at which significant changes in the trends occur. For every time period, the linear slope of the trend, p value, observed number of deaths, CHD mortality rates and change in the CHD mortality rate were calculated. RESULTS: Between 1972 and 2007, the age-adjusted CHD mortality rates decreased overall by 76% in both men and women. In men (35-54 years), the change in CHD mortality rate in the period 1980-1993 was -0.53 but attenuated in period 1993-1999: -0.16. In women (35-54 years) the decline likewise attenuated to -0.44 in period 1979-1989: and -0.05 in period 1989-2000. After 1999-2000, CHD mortality rate further declined in both men (period 1999-2007: -0.46) and women (period 2000-2007: -0.38). CONCLUSIONS: Evidence from several Western countries suggests that among young adults (< 55 years), CHD mortality rates are levelling out. In this study, similar attenuation of the decline in CHD mortality among young adults in the Netherlands has been observed. Furthermore, this is the first study to observe a subsequent increase in the pace of decline after a period of flattening. In order to better explain these encouraging changes in CHD mortality rates, a detailed analysis of recent changes in cardiovascular risk factors and treatments is now urgently required.

Coronary heart disease trends in England and Wales from 1984 to 2004: concealed levelling of mortality rates among young adults.

BACKGROUND: Trends in cardiovascular risk factors among UK adults present a complex picture. Ominous increases in obesity and diabetes among young adults raise concerns about subsequent coronary heart disease (CHD) mortality rates in this group. OBJECTIVE: To examine recent trends in age-specific mortality rates from CHD, particularly those among younger adults. METHODS AND RESULTS: Mortality data from 1984 to 2004 were used to calculate age-specific mortality rates for British adults aged 35+ years, and joinpoint regression was used to assess changes in trends. Overall, the age-adjusted mortality rate decreased by 54.7% in men and by 48.3% in women. However, among men aged 35-44 years, CHD mortality rates in 2002 increased for the first time in over two decades. Furthermore, the recent declines in CHD mortality rates seem to be slowing in both men and women aged 45-54. Among older adults, however, mortality rates continued to decrease steadily throughout the period. CONCLUSIONS: The flattening mortality rates for CHD among younger adults may represent a sentinel event. Deteriorations in medical management of CHD appear implausible. Thus, unfavourable trends in risk factors for CHD, specifically obesity and diabetes, provide the most likely explanation for the observed trends.

Progresión de la poliquistosis renal autosómica dominante. Influencia de polimorfismos de genes de sintasa endotelial del óxido nítrico (ecNOS) y del sistema renina-angiotensina / Progression of autosomic dominant polycystic kidney disease. Influence of endothelial NO synthase (ecNOS) and renin angiotensin system gene polymorphisms

Glomerular filtration rate decline (GFRd) is variable in autosomic dominant polycystic kidney disease (ADPKD). In 88 ADPKD patients, GFRd was assessed by 1/S(Cr) and compared with the association to AT1A1166C (AT1R), AGTM235T (angiotensinogen) and ecNOSGlu298Asp (NO endothelial synthase) polymorphisms. Age at S(Cr) values of 2 and 6 mg/dl were assumed as beginning of progressive phase (A2) and end-stage-renal disease (A6), respectively. Polymorphisms were studied by PCR-RFLP. The group as a whole showed GFRd (ml/min/year) of 6.9+/-0.5; A2 and A6 of 48.9+/-1.3 and 55.0+/-1.4 years and mean arterial pressure of 111.2+/-1.2 mmHg. When A6 was considered, two populations were defined (< or = and > 55 years). In < or = 55 (assumed as PKD1 phenotype) (n=42), A2 and A6 of the AT1 1166CC genotype were 36.0+/-1.2 and 41.4+/-0.9 years vs AA-AC (42.8+/-1.0 and 47.5+/-0.8, p<0.001). A2 and A6 of the ecNOS298Asp/Asp genotype were 34.8+/-1.5 and 41.1+/-0.6 years vs. Glu/Glu-Glu/Asp (42.4+/-0.9 and 47.1+/-0.8, p<0.02). In AGT235TT genotype, GFRd was 12.4+/-2.2 ml/min/year vs MM-MT (7.9+/-0.7, p<0.03). This difference was also observed when all ADPKD patients were considered (TT: 11.02+/-1.5 vs. MM-MT: 6.44+/-0.5 ml/ min/year, p<0.003). AT1 1166CC and ecNOS 298Asp/Asp are associated with earlier A2 and A6 whereas AGT 235TT induce twofold increase in GFRd, suggesting that RAS and ecNOS are involved in ADPKD progression.

La velocidad de progresión (VdP) de la poliquistosis renal autosómica dominante (PQRAD) es variable.Estudiamos la asociación de los polimorfismos AGTM235T (angiotensinógeno), AT1A1166C(ATR1) y ecNOSGlu298Asp (NO sintasa endotelial) con la VdP en 88 pacientes. VdP fue estimada por 1/Crplvs edad. Consideramos edades de Crpl 2 y 6 mg/dl como comienzo de progresión (E2) y arribo a insuficienciarenal crónica terminal (E6), respectivamente. Los polimorfismos se estudiaron por PCR-RFLP. El grupo en sutotalidad presentó VdP (ml/min/año) de 6.9±0.5, E2 y E6 de 48.9±1.3 y 55.0±1.4 años y tensión arterial media(TAM) de 111.2±1.2 mmHg. Según E6 observamos dos grupos (≤ y > a 55 años). En ≤ 55 (fenotipo PKD1,n=42), E2 y E6 del genotipo CC de AT1A1166C fueron 36.0±1.2 y 41.4±0.9 años vs. AA-AC (42.8±1.0 y 47.5±0.8, p < 0.001). E2 y E6 del genotipo ecNOS298Asp/Asp fueron 34.8±1.5 y 41.1±0.6 años vs. Glu/Glu-Glu/Asp (42.4±0.9 y 47.1±0.8, p < 0.02). En el genotipo AGT235TT, la VdP fue 12.4±2.2 ml/min/año vs. MM-MT (7.9±0.7, p < 0.03). Esta diferencia también se observó cuando analizamos todos los pacientes PQRAD (TT: 11.02±1.5 vs. MM-MT: 6.44±0.5 ml/min/año, p < 0.003). Los genotipos AT1 1166CC y ecNOS 298Asp/Asp anticipan E2 y E6 mientras que AGT235TT duplica VdP, sugiriendo la participación del sistema renina angiotensina y NO sintasaendotelial en la progresión de la PQRAD.

Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens.

The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth-inhibitory activity towards the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin-1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin-2 family, first identified in the European frog Rana esculenta; the ranatuerin-2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin-2, ranalexin, esculentin-1 and ranatuerin-1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino-acid sequences of the antimicrobial peptides produced by each species are highly variable and species-specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.

[Epidemiology of delirium in elderly inpatients]. / Epidemiología del delirio en ancianos hospitalizados.

Our objective was to determine delirium incidence and risk factors in a cohort of elderly inpatients. We randomly selected 149 patients, aged 65 years or older, from admission to general wards, without evidence of delirium. They were evaluated daily with the Confusion Assessment Method, an instrument validated for the diagnosis of delirium. We obtained relative risks for delirium and those independently associated were included in a logistic regression model. We used the chi-square test with Yate's corrections for univariate analysis, and t-test for comparisons of means. We observed that 51 patients (20.5%) developed delirium during their hospital stay. Severity of disease (RR 1.28, 1.14-1.43), having chronic diseases (RR 3.45, 2.4-4.96), and having fever at admission (RR 1.84, 1.33-2.56) were found independently associated with delirium. Patients who developed delirium had longer hospital stay (9.87 days +/- 3.48 vs 6.95 days +/- 2.45, p < 0.05) and higher mortality (RR 2.19, CI 1.26-3.79). We conclude that delirium in our setting is very frequent and has negative effects on resource utilization and mortality in elderly inpatients. Its association with the severity of the disease seems interesting. Appropriate prospective identification of patients at risk for delirium may allow the implementation of preventive strategies in order to minimize the impact of this complication.

Effect of accessible immobilized NAD(+) concentration on the bioaffinity chromatographic behavior of NAD(+)-dependent dehydrogenases using the kinetic locking-on strategy.

In preparation for studies aimed at establishing the relationship between immobilized NAD(+) concentration and the concentration of soluble locking-on ligand required to promote biospecific adsorption of NAD(+)-dependent dehydrogenases to immobilized NAD(+) derivatives (the "locking-on" strategy), two approaches were evaluated for varying substitution levels: (i) suitable dilution of the affinity matrix with unsubstituted Sepharose 4B and (ii) direct coupling of the required ligand concentration to the inert matrix. The latter approach was found to be the preferable strategy for evaluation of the locking-on tactic because it produced a more homogeneous distribution of immobilized NAD(+) concentration. Affinity chromatographic studies using S(6)-linked NAD(+) derivatives synthesized to various substitution levels showed that the total accessible immobilized NAD(+) concentration has a direct effect on the locking-on behavior of pyridine nucleotide-dependent dehydrogenases. The one-chromatographic-step bioaffinity purification of l-lactate dehydrogenase (L-LDH, EC 1.1.1.27) from bovine heart illustrates the potential of the locking-on strategy for protein purification applications.

The kinetic locking-on strategy for bioaffinity purification: further studies with bovine liver glutamate dehydrogenase.

The locking-on strategy uses soluble analogues of the enzymes specific substrate to produce biospecific adsorption of individual NAD(P)(+)-dependent dehydrogenases on immobilized NAD(P)(+) derivatives, which is so selective that a single enzyme activity can be purified from crude cellular extracts in a single chromatographic step with yields approaching 100%. However, attempts to further develop and apply this strategy to the biospecific chromatographic purification of a range of NAD(P)(+)-dependent dehydrogenases revealed some anomalous chromatographic behavior and certain unexplained phenomenon. Much of this can be attributed to nonbiospecific interference effects. Identification and elimination of this interference is discussed in the present study focusing on bovine liver glutamate dehydrogenase (GDH; EC 1.4.1.3) as the "test" enzyme. Results further confirm the potential of the locking-on strategy for the rapid purification of NAD(P)(+)-dependent dehydrogenases and provide further insight into the parameters which should be considered during the development of a truly biospecific affinity chromatographic system based on the locking-on strategy. The kinetic mechanism of bovine liver GDH has been the topic of much controversy with some reports advocating a sequential ordered mechanism of substrate binding and others reporting a sequential random mechanism. Since the kinetic locking-on strategy is dependent on the target NAD(P)(+)-dependent dehydrogenase having an ordered sequential mechanism of substrate binding, the bioaffinity chromatographic behavior of bovine liver GDH using the locking-on tactic suggests that this enzyme has an ordered sequential mechanism of substrate binding under a variety of experimental conditions when NAD(+) is used as cofactor.

A "stripping" ligand tactic for use with the kinetic locking-on strategy: its use in the resolution and bioaffinity chromatographic purification of NAD(+)-dependent dehydrogenases.

The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD(+)-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD(+)-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously "dribbled" off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5'-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD(+)-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (l-LDH, EC 1.1.1.27).