Abstract:

Abstract: Life-long antigenic burden determines a condition of chronic inflammation, with increased lymphocyte activation and proinflammatory cytokine production. A large number of studies have documented changes in zinc metabolism in experimental animal models of acute and chronic inflammation and in human chronic inflammatory conditions. In particular, modification of zinc plasma concentration, as well as intracellular disturbance of antioxidant intracellular pathways, has been found in aging and in some age-related diseases. Zinc deficiency is diffused in aged individuals in order to avoid meat and other high zinc content foods due to fear of cholesterol. Rather, they increase the consumption of refined wheat products that lack zinc and other critical nutrients as a consequence of the refining process. On the other hand, plasma zinc concentration is influenced by proinflammatory cytokines (IL-6 and TNF-alpha) and by metallothioneins (MT) homeostasis, which is in turn affected by proinflammatory cytokines. MT increase in aging and chronic inflammation allowing a continuous sequestration of intracellular zinc with subsequent low zinc ion availability against stressor agents and inflammation. This phenomenon leads to an impaired inflammatory/immune response in the elderly. A major target of zinc is NF-kappaB, a transcription factor critical for the expression of proinflammatory cytokines whose production is regulated by extra- and intracellular activating and inhibiting factors interacting with the regulatory elements on cytokine genes. Effects of zinc on translocation of NF-kappaB have been attributed to the suppression of phosphorylation and degradation of the inhibitory proteins (A20) that normally sequester it in the cytoplasm. Moreover, this factor and A20 are regulated by specific genes involved in inflammation and by intracellular zinc ion availability. So, it is not so surprising that zinc deficiency is constantly observed in chronic inflammation, such as in old individuals. On the other hand, cytokine genes are highly polymorphic and some of these polymorphisms are associated with atherosclerosis and diabetes type 2. Therefore, zinc turnover, via MT homeostasis, in individuals genetically predisposed to a dysregulation of the inflammatory/immune response may play a crucial role in causing possible adverse events with the appearance of age-related diseases.

Abstract: The study of the interactions among biological factors and psychosocial conditions is a very innovative field, because data are lacking in the scientific literature. Among biological aspects, zinc is an essential element in the elderly, especially in relation to one of the proteins, such as albumin, involved in zinc transport into the cells. In this study, the aim is the assessment of the interrelationship between albumin value (used as an index of the body zinc status) and some psychosocial dimensions in elderly Italian sample recruited for ZINCAGE project, supported by the European Commission in the "Sixth Framework Programme". Some tests and questionnaires were administered to older people included in the trial: the "life-style questionnaire"; the mini mental state examination (MMSE); the geriatric depression scale (GDS-15 items). On the basis of the Senieur Protocol for gerontological studies, a sample of 291 Italian healthy old subjects has been recruited in Central Italy and divided into 3 age groups: (a) 125 subjects aged from 65 to 74 years, (b) 89 subjects aged from 75 to 84 years, (c) 77 subjects aged >or=85 years (classified like successful old people). No cognitive impairment assessed by MMSE was observed in 67.5% of the sample; 64.0% had GDS score less than 5, indicating no depression, whereas the prevalence of biological albumin deficiency (<3.5 g/dl) found in Italian old people was 21.0%. Sixty one percent of subjects with albumin deficiency displayed higher values of GDS (>or=5). These preliminary results showed an interrelationship among serum albumin value and psychosocial aspects in Italian old population, suggesting that low albumin values may be involved in impaired psychological dimensions.

Abstract: ABSTRACT: The relevance of zinc for proper functioning of the entire immune system is already well documented. However, the identification of individuals who really need zinc supplementation is still debated in view of the fact that excessive zinc may also be toxic. The risk of developing zinc deficiency in people from industrialized countries is relatively low, except for elderly subjects where zinc intake may be suboptimal and inflammation is chronic. Thus, the role of zinc on the immune system and on the health of European elderly people is becoming of paramount importance, considering also that the elderly population is rapidly increasing. In particular, the factors contributing to and the biochemical markers of zinc deficiency in the elderly are still remain to be established. Epidemiological, functional, and genetic studies aimed at formulating a rationale for the promotion of healthy ageing through zinc supplementation was the subject of an International Conference held in Madrid from 11-13 February 2006 (3rd ZincAge Meeting) at the CNIO Institute (local organizer: Maria Blasco, partner of ZincAge).

Abstract: Thymic re-growth and reactivation of thymic functions may be achieved in old animals by different endocrinological or nutritional manipulations such as, (a) treatment with melatonin, (b) implantation of a growth hormone (GH) secreting tumour cell line (GH3 cells) or treatment with exogenous GH, (c) castration or treatment with exogenous luteinizing hormone-releasing hormone (LHRH), (d) treatment with exogenous thyroxin or triiodothyronine, and (e) nutritional interventions such as arginine or zinc supplementation. These data strongly suggest that thymic involution is a phenomenon secondary to age-related alterations in neuroendocrine-thymus interactions and that it is the disruption of these interactions in old age that is responsible for age-associated immune-neuroendocrine dysfunctions. The targets involved in hormones-induced thymic reconstitution may directly or indirectly involve hormone receptors, cytokines, arginine, and a trace element such as zinc, which is pivotal for the efficiency of neuroendocrine-immune network during the whole life of an organism. The effect of GH, thyroid hormones, and LHRH may be due to specific hormone receptors on thymocytes and on thymic epithelial cells (TECs), which synthesize thymic peptides. Melatonin may also act through specific receptors on T-cells. In this context, the role of zinc, which turnover is reduced in old age, is pivotal because of its involvement through zinc fingers in the gene expression of hormone receptors. In addition, the effects of zinc are multifaceted: from the reactivation of zinc-dependent enzymes, to cell proliferation and apoptosis, to cytokines expression and to the reactivation of thymulin, which is a zinc-dependent thymic hormone required for intrathymic T-cell differentiation and maturation as well as for the homing of stem cells into the thymus. Zinc is also required for arginine action, via NO pathway. The role of zinc is therefore crucial in neuroendocrine-thymus interactions. According to data in animals and humans, the above reported endocrinological manipulations (GH, thyroid hormones, and melatonin) or arginine treatment may also act via zinc pool in restoring thymic activity in ageing allowing improvements on peripheral immune efficiency.

Abstract: Proteins involved in zinc homeostasis may be altered in aging. This phenomenon may lead to zinc deficiency in the peripheral blood and an accumulation of zinc bound to insoluble aggregates at the extracellular level in the brain. Therefore, it should be more correct to talk about aging as a condition associated with zinc dyshomeostasis rather than deficiency. Restoring functional zinc homeostasis in aging people is an attractive field for antiaging research, but requires further knowledge than the current state of the art.

Abstract: Brain performances, e.g. learning and memory, decay during aging. Deterioration of synaptic junctions, as structural correlates of these key functions of the central nervous system, may play a central role in this impairment. Current research on the age-related changes of synapses is documenting that the numeric loss of contacts appears to trigger a compensatory reaction by the old CNS, i.e. the surviving junctional areas in old individuals are larger than in adult subjects. The final outcome of the balanced changes in synaptic number and size is that the overall synaptic junctional area per cubic micron of neuropil is also reduced in aging and this may account for the age-associated functional decay of CNS performances. Among the suggested determinants of synaptic deterioration in aging, a considerable number of recent studies support an early and pivotal role of the progressive decline of the mitochondrial metabolic competence, i.e. the capacity of select pools of organelles to provide adequate amounts of adenosine triphosphate. Quantitative ultrastructural studies together with cytochemistry of key enzymes of the respiratory chain (cytochrome oxidase and succinic dehydrogenase) have shown that mitochondrial dysfunctions play an early and central role in synaptic deterioration events associated with aging and neurodegenerative diseases. Among the various causes, the multiple mechanisms and molecules involved in zinc ion homeostasis have been supposed to be less efficient in the aging brain. Thus, a transient imbalance of free zinc ion concentration in the cytosol ([Zn2+]i) can be considered an unfavourable trigger of subtle mitochondrial damage and synaptic pathology.

Abstract: ABSTRACT : Longitudinal studies are defining progressive alterations to the immune system associated with increased mortality in the very elderly. Many of these changes are exacerbated by or even caused by chronic T cell stimulation by persistent antigen, particularly from Cytomegalovirus. The composition of T cell subsets, their functional integrity and representation in the repertoire are all markedly influenced by age and by CMV. How these findings relate to epidemiological, functional, genetic, genomic and proteomic studies of human T cell immunosenescence was the subject of intense debate at an international conference held just before Christmas 2005 in the Black Forest.

Abstract: Coronary artery disease (CAD) is characterized by an inflammatory status and it represents the major cause of death in elderly. Zinc deficiency and inflammatory genes within major histocompatibility complex (MHC) region are implicated in ischaemic heart diseases. TNF-alpha is present in coronary artery plaques and may provoke plaque instability. Hsp70 plays instead a pro-atherogenic role, via proinflammatory cytokine production, in atherosclerotic lesions contributing to plaque rupture. Contradictory data report the association between -308 TNF-alpha polymorphism and CAD, while no investigations exist on Hsp70-2 gene in CAD. In the current study, we analysed -308 TNF-alpha and 1267 Hsp70-2 polymorphisms and zinc status in 190 healthy old controls and 216 old patients with carotid stenosis subdivided in two groups: the first one 105 patients with CAD (C group), and the second one 111 patients without cardiovascular events (D group). We found a lack of association between -308 TNF-alpha polymorphism and CAD. Conversely, 1267 Hsp70-2 polymorphism was associated with CAD. In particular, significant higher frequency of AB + BB genotypes (B + genotype) was observed in C patients than controls (71.4 vs.56.9%, P = 0.017, odds ratio = 1.898). However, when C patients were subdivided into four subgroups on the basis of presence/absence of 1267B Hsp70-2 and -308A TNF-alpha alleles, B + A + patients showed higher prothrombin activity as well as Hsp70-2, TNF-alpha, IL-6 gene expressions in carotid atheroma when compared to B - A - genotypes. The zinc status (plasma and Zn/Fe ratio in erythrocytes) is not affected by these polymorphisms. However, zinc deficiency is present in CAD condition. In conclusion, 1267 HSP70-2 polymorphism and zinc deficiency, rather than -308 TNF-alpha, are independently associated with CAD. B + A+ and B + A- carriers seem more predisposed to ischaemic events; conversely, B - A- genotype may be considered a protective marker against CAD.

Abstract: It is very interesting and innovative to study the interrelationships between biological characteristics, particularly zinc status, and psychosocial conditions in old age, because there are few and fragmentary data in the literature. The aim of this study was to examine the interrelationship between serum albumin value (an indicator of zinc status) and some psychosocial characteristics in elderly Italian volunteers recruited for the ZINCAGE project, which is supported by the European Commission in the Sixth Framework Programme (Food-CT-2003- 506850). A protocol of tests and questionnaires was used: the Lifestyle Questionnaire, the Mini-Mental Status Examination (MMSE), the Geriatric Depression Scale (GDS, 15 items), and the Perceived Stress Scale. A sample of 174 old subjects were recruited in Region Marche (Central Italy), and classified into three age groups: 65 to 74 years old, 75 to 84 years old, and >85 years old (including some nonagenarians). The preliminary results show that 69.7% of the subjects have no cognitive impairment; 66.5% have a value of the GDS scale indicating no depression; and 17% have an albumin deficiency. The majority of these are >85 years old and women. A relationship between level of albumin (used as indicator of zinc status) and depression has been found: 71% of subjects with albumin deficiency displayed a higher value on the depression test against 29% of subjects with a normal value (p < 0.01). These preliminary results show a relationship between serum albumin and psychological characteristics, in particular depression in an old Italian population. This further suggests that a zinc deficiency, via hypoalbumin values, is involved in impaired psychological characteristics in the elderly.

Abstract: In this paper, we reviewed data regarding to the pivotal role played by the zinc-gene interaction in affecting some relevant cytokines (IL-6 and TNF-alpha) and heat shock proteins (Hsp70-2) in ageing, successful ageing (nonagenarians) and in some age-related diseases (atherosclerosis and infections). The polymorphisms of the genes codifying these proteins are predictive on one hand in longevity, such as IL-6 -174G/C locus, on the other hand 1267 Hsp70-2A/B or TNF-alpha -308G/A polymorphisms are associated to worsening atherosclerosis or severe infections, respectively, rather than longevity. Taking into account that longevity has a strong genetic component but, at the same time, is affected by life style and environmental factors, the analysis of these polymorphisms in association to some immune parameters (NK cell cytotoxicity) and nutritional factors (zinc) is a useful tool to unravel the role played by these genetic factors in longevity and in the appearance of age-related diseases. Indeed, these polymorphisms are associated with chronic inflammation, low zinc ion bioavailability, depressed innate immune response and high gene expression of metallothioneins, which have a limited zinc release for an optimal innate immune response in ageing. Therefore, the nutrient (zinc)-gene (IL-6, TNF-alpha and Hsp70-2) interaction is pivotal to keep under control the inflammatory/immune response with subsequent longevity, indicating these genes as "robust" for "healthy ageing".

Abstract: BACKGROUND:: The amount of available intracellular zinc is pivotal to regulate many cellular processes, including oxidative stress response and apoptotic mechanisms. Therefore it is not surprising that zinc homeostasis and dyshomeostasis is involved in many physiological and pathological states, respectively. Cell permeable zinc probes allow intracellular applications with microscopy technology, but flow cytometry (FC) applications have been scarcely explored, albeit they can be suited to study zinc homeostasis in different cell types, including rare cells. METHODS:: We describe a FC method able to estimate intracellular zinc ion availability and the intracellular capability to activate a zinc signal after treatment with an NO-donor (AcOM-DEA/NO) in human PBMCs, using the fluorescent zinc-specific probe, Zinpyr-1 (ZP1), alone or in association with CD4-PE and CD8-Cychrome mAb. RESULTS:: This method was able to detect an increase/decrease of intracellular zinc available in human fresh cultured PBMC and in immune subsets using AcOM-DEA/NO or TPEN, respectively. ZP1 mean fluorescence on gated histograms was sensitive to the amount of zinc added in the culture medium and significantly correlated to metallothioneins and total intracellular zinc. CONCLUSIONS:: FC applications using ZP1 may be a fast and useful tool to study zinc homeostasis in immune cells.

Abstract: Zinc is a relevant trace element for the efficiency of the entire immune system. The binding of zinc with some proteins, such as metallothioneins (MT) and alpha-2 macroglobulin (alpha-2M) is crucial for the immune efficiency during ageing and in age-related diseases, because these proteins may be involved in antagonistic pleiotropic effects. Indeed, the presence of chronic inflammation during ageing, generally, induces overexpression of these proteins that, due to their original biological function in fighting stressor agents, continuously sequester intracellular zinc. As a consequence, a low zinc ion availability may appear in aged organisms leading to impairments of the immune response at thymic and extrathymic levels with the risk of the appearance of age-related diseases. Therefore, MT and alpha-2M turn from protective in "young-adult age" to harmful agents in "ageing" following the basic assumption of an evolutionary theory of ageing, named the "antagonistic pleiotropy", which suggests that a trade off between early beneficial effects and late negative outcomes can occur at a genetic and molecular level. On the other hand, some polymorphisms of MT (MT2A) and alpha-2M have been associated with atherosclerosis or Alzheimer disease, respectively. Physiological zinc supplementation in elderly restores the thymic endocrine activity and innate immune response (NK cell cytotoxicity) and increases the survival rate in old mice. Therefore, zinc supplementation is useful to achieve health longevity because these zinc-binding proteins may regain their original protective task against oxidative damage with, thus, a beneficial impact on immune response.

Abstract: As the elderly population is increasing rapidly, there is a lot of scientific interest in clarifying the differential life-style, genetic, biochemical and molecular factors contributing to mortality or exceptional longevity. Within the framework of the ZINCAGE project, 249 old (60-85 years) and nonagenarian Greek subjects (>/=85 years old) were recruited and anthropometrical, blood and biochemical indices as well as blood pressure measurements were obtained. Based upon the inclusion criteria, 214 of them were characterized as healthy (136 female, 78 male), while those characterized as non-healthy were excluded from the statistical analysis. The findings indicated an 88.4% prevalence of overweight and obesity in elderly (91.2% in women, 83.3% in men), while a significant decrease in body mass index with age was recorded. Hypercholesterolemia was observed in 75.3% of elderly women and in 69.2% of men. Erythrocyte sedimentation rate value was increased in 27.6% of all healthy elderly, while zinc deficiency was observed in 18.7% of elderly. Over 85.0% of all healthy elderly were within normal range for all other hematological analysis. Blood analysis confirmed the good health status of the elderly recruited as healthy, except for the high prevalence of obesity and hypercholesterolemia.

Abstract: BACKGROUND: CD14 receptor is a mediator of the inflammatory response to bacterial products. A functional polymorphism in the promoter of the CD14 gene (CD14 C-260T) was associated with coronary heart disease and atherosclerosis albeit with conflicting data. METHODS: To better clarify the role of CD14 in atherosclerosis, we typed CD14 C-260T polymorphism in old Italian (Central of Italy) atherosclerotic patients with carotid stenosis related to lipid assessment, inflammation (soluble CD14, IL-6 serum levels) and IL-6, TNF-alpha, IL-10, Metallothioneins (MT) gene expressions in carotid plaques. RESULTS: There was an increased frequency of TT homozygotes in patients when compared to controls [26% vs. 13.5%, odds ratio=2.25 (95% C.I., 1.23-4.09, p=0.0082) (Fisher's Exact test)]. Subjects with TT genotype showed a significant increase of soluble CD14 and enhanced MT2A, IL-6, TNF-alpha and decreased IL-10 gene expressions within the carotid plaques. On the basis of lipid assessment, hypercholesterolemic -260TT CD14 patients displayed lower HDL cholesterol and higher triglyceride than did CT and CC carriers. Using Pearson's correlation, a high MT2A expression was associated with high IL-6, TNF-alpha, sCD14 and thereby with severe chronic inflammation. CONCLUSIONS: These data provide insight into the pathogenetic role of the CD14 C-260T polymorphism in atherosclerosis as -260TT genotype may favour increased inflammation in atheroma promoting possible worsening atherosclerosis, at least in Central of Italy elderly population. Further studies are in progress in cohorts from different European geographic area (Zincage project).

Abstract: Lifelong antigenic burden determines a condition of chronic inflammation, with increased lymphocyte activation and pro-inflammatory cytokine production. A large number of studies have documented changes in Zn metabolism in experimental animal models of acute and chronic inflammation and in human chronic inflammatory diseases. In particular, modification of zinc plasma concentration as well as intracellular disturbance of antioxidant intracellular pathways have been found associated to age-related inflammatory diseases, like atherosclerosis. Zinc deficiency is extremely diffused in aged people that are educated to avoid meat and other high Zn-content foods due to fear of cholesterol. Rather, they increase consumption of refined wheat products that lack of Zn, magnesium and other critical nutrients in consequence of refining process. On the other hand, plasma concentration of metallic ions like Zn is influenced by pro-inflammatory cytokines production. A major target of Zn may be NF-kB, a transcription factor critical for the expression of many pro-inflammatory cytokines whose production is finely regulated by extra- and intracellular activating and inhibiting factors interacting with regulatory elements on cytokine genes. Moreover, this factor is regulated by the expression of specific cellular genes involved in inflammation. So it is not surprising that Zn deficiency is constantly observed in aged patients affected by infectious diseases. On the other hand, cytokine genes are highly polymorphic and some of these polymorphisms have been found associated to age-related diseases as atherosclerosis. Therefore, Zn deficiency in individuals genetically predisposed to a dis-regulation of inflammation response, may play a crucial role, in causing adverse events and in reducing the probability of a successful aging.

Abstract: Metallothioneins (MTs) play a pivotal role in zinc-related cell homeostasis because of their high affinity for zinc, which is in turn fundamental for immune response and antioxidant activity. MTs regulate zinc homeostasis by binding zinc and releasing zinc at the occurrence for immune response. The zinc release by MT is very limited in chronic inflammation and ageing. Some polymorphisms of MTs gene, in particular MT1a sub-isoform, may affect this release that is a problem still unresolved in ageing. The screening in the present paper of two polymorphisms in MT1a gene has revealed for the first time that the polymorphism corresponding to a A/C (Asp/Thr) transition at 647 nt position in the Mt1a coding region is the more involved in the longevity, at least in old women, rather than the other corresponding to A/G (Lys/Arg) transition at 1,245 nt position. Concomitantly, for the +647 MT1a polymorphism, old and very old female with Asp/Asp genotype (called C-carriers) display higher zinc release by MT (detected by Zinpyr-1 fluorescent probe in presence of NO donor), low MT levels and reduced IL-6 plasma concentrations, suggesting its involvement in longevity and in lower inflammatory status. This fact is confirmed by the analysis of haplotypes in which the allele Asp is more involved in longevity. Therefore the +647 MT1a polymorphism more affects MTs induction and zinc release, which are indispensable to undertake the inflammatory status under control and subsequently to reach healthy longevity.

Abstract: Cellular components of both adaptive and innate immune systems produce different chemokines and cytokines, involved in different signalling pathways among cells, and modulate effector function during immune response, playing a key role in the regulation of the type and extent of the immune response in the elderly. We evaluated the circulating concentration of selected chemokines: MCP-1, MIP-1alpha, IL-8, RANTES together with IL-6 and TNF-alpha in plasma obtained from a group of healthy old subjects, in order to highlight possible differences in the synthesis of these factors, assuming that both the cytokine and the chemokine networks are remodelled with ageing. The simultaneous evaluation was performed by a multiplex analysis system. In addition, since micronutrient deficiency may underlie an inflammatory response, the association between chemokine levels and a nutritional element such as zinc was also evaluated, since the immune system is the first system to be affected by changing zinc levels, due to its high cell turnover. A progressive age-related increase of plasma concentrations of all soluble factors was observed. The increment was particularly evident for IL-6, IL-8, MCP-1 and TNF-alpha in the over 85-year-old group in concomitance with increasing percentages of subjects with low circulating levels of zinc. In conclusion, the remodelling of chemokine profiles, skewed to Th2 response by both advanced ages and circulating levels of zinc, might reflect different states of activation and/or responsiveness of the human immune cell/mediator network, thus influencing the ability to develop rapid innate and long-lasting adaptive immune responses with advancing age.

Abstract: Mild zinc deficiency is a common condition in healthy elderly individuals leading to impaired cell-mediated immune response. Here we report the effect of improved zinc status on TH1/TH2 balance and on the activation status of T helper cells in 19 healthy elderly subjects aged 69.8 +/- 5.1 years. Our investigations revealed a mild zinc deficiency which was adjusted by oral zinc supplementation for seven weeks. Improved serum zinc levels significantly reduced levels of activated T helper cells whereas changes in TH1/TH2 ratio (determined by CCR4 and CCR5 expression) were not observed. These findings suggest that elderly individuals may benefit from moderate zinc supplementation due to improved immune response leading to reduced incidences of autoimmune diseases and infections.

Abstract: Enzymatic activities of plasma superoxide dismutase (pSOD), catalase (CAT) and glutathione peroxidase (GPx) and erythrocyte superoxide dismutase (eSOD) were assayed in 981 healthy community dwelling old subjects participating in the Zincage Project. The relationship between antioxidant enzyme activities and, respectively, gender, age and zinc status were assessed. eSOD activity was higher in nonagenarians than in 80 year old subjects. Plasma Zn was lower in nonagenarians compared with younger subjects. The prevalence of Zn deficiency increased with age, with normal Zn levels observed in about 80% of adult subjects and only in 37% of the nonagenarians. Women showed higher eSOD and CAT activities compared to men, whereas plasma Zn was higher in men than in women. There was a positive correlation between eSOD activity and age and a negative correlation between eSOD activity and plasma Zn concentrations. An inverse correlation was also found between plasma Zn concentration and age. Further studies on different aspects of Zn metabolism-intake, plasma concentration, peripheral cell concentration, activity and amount of Zn-dependent enzymes-are warranted.

Abstract: Actual fields of research in neurobiology are not only aimed at understanding the different aspects of brain aging but also at developing strategies useful to preserve brain compensatory capacity and to prevent the onset of neurodegenerative diseases. Consistent with this trend much attention has been addressed to zinc metabolism. In fact, zinc acts as a neuromodulator at excitatory synapses and has a considerable role in the stress response and in the functionality of zinc-dependent enzymes contributing to maintaining brain compensatory capacity. In particular, the mechanisms that modulate the free zinc pool are pivotal for safeguarding brain health and performance. Alterations in zinc homeostasis have been reported in Parkinson's and Alzheimer's disease as well as in transient forebrain ischemia, seizures and traumatic brain injury, but little is known regarding aged brain. There is much evidence that that age-related changes, frequently associated to a decline in brain functions and impaired cognitive performances, could be related to dysfunctions affecting the intracellular zinc ion availability. A general agreement emerges from studies of humans' and rodents' old brains about an increased expression of metallothionein (MT) isoforms I and II, but dyshomogenous results are reported for MT-III, and it is still uncertain whether these proteins maintain in aging the protective role, as it occurs in adult/young age. At the same time, there is considerable evidence that amyloid-beta deposition in Alzheimer's disease is induced by zinc, but the pathological significance and the causes of this phenomenon are still an open question. The scientific debate on the role of zinc and of some zinc-binding proteins in aging and neurodegenerative disorders, as well as on the beneficial effect of zinc supplementation in aged brain and neurodegeneration, is extensively discussed in this review.

Abstract: Patients with type 2 diabetes mellitus (NIDDM) are at risk for macrovascular disease complications, such as myocardial infarction (MI) or stroke from plaque rupture. Cytokines play a key role in plaque vulnerability. IFN-gamma inhibits collagen synthesis thereby affecting plaque stability. High IL-6, TNF-alpha, and dyslipidemia are risk factors for thrombosis. Abnormal increments of HSP70 in atherosclerotic plaques might lead to plaque instability and rupture caused by chronic inflammation, which up-regulates the expression of pro-inflammatory cytokines (IL-6 and TNF-alpha) in human monocytes. Studies of a polymorphic PstI site lying in the coding region at position 1267 of the HSP70-2 gene have shown that the BB genotype is associated with NIDDM. We screened 60 old NIDDM patients with carotid stenosis and 107 old healthy controls for 1267 HSP70-2 polymorphism in order to establish if an association with plaque frailty exists. Different genotypic distributions were observed between patients and healthy controls. An increased relative risk was associated with the B allele (p = 0.0107; odds ratio = 1.861). HSP70-2, IL-6, IFN-gamma, TNF-alpha gene expressions within the plaques and serum levels of triglyceride, total cholesterol and LDL cholesterol were tested from patients stratified according to their B+ (AB and BB) and B- (AA) genotypes. Plaque morphology (soft or fibrous-calcified) and the incidence of cerebral ischaemia were also assessed. B+ patients showed increased HSP70-2, IL-6, IFN-gamma, TNF-alpha and dyslipidemia as compared to B- carriers. The frequency of soft plaques increased in B+ in comparison to B- patients (67% versus 13%; odds ratio 13.0, p = 0.0006). A higher frequency of cerebral ischaemia (ictus or transient ischaemic attack (TIA)) was present in B+ than in B- genotype (53% versus 20%; odds ratio 4.57, p < 0.05) Hence, 1267 HSP70-2 polymorphism may be of use in identifying B+ NIDDM patients at risk for carotid plaque rupture and cerebral ischaemia.

Abstract: Abnormal increments of pro-inflammatory cytokines (IL-6 and TNF-alpha) characterize the outbreak of infectious diseases, which are the major cause of death in the elderly. A counterbalance to the inflammation is exerted by IL-10 with an inhibitory role on TNF-alpha production. As is well known, some cytokine gene polymorphisms influence the cytokine production, playing a role as susceptibility or resistance factors against immune-mediated and infectious disease. Genetic variations in the -308A/G locus for TNF-alpha seems to affect the clinical outcome of some infectious diseases. In fact, the -308A allele is associated with severe septic shock and death. On this basis, we have screened healthy old subjects, nonagenarians and old patients affected by the acute phase of chronic obstructive bronchitis and bronchopneumonia of bacteria origin for the -308G/A locus (PCR-RFLP). Subjects are grouped in A+ (AG, AA genotypes) and A- (GG genotype) and data on IL-6, TNF-alpha, IL-10, NK cell cytotoxicity, zinc and metallothioneins (MTs) gene expression (RT-PCR) were stratified according to different TNF-alpha genotypes. The frequency of the A allele was increased in infected patients in comparison with healthy old controls. No differences existed between A+ and A- young adult, old and nonagenarian controls in tested parameters. Conversely, A+-infected patients displayed elevated IL-6, TNF-alpha and MTmRNA, low IL-10 coupled with impaired NK cell cytotoxicity and lower zinc ion than A- patients. However, the data reported are gender independent. Therefore, the -308A polymorphism at the locus of TNF-alpha may be one of the susceptibility factor for infectious diseases in old persons, particularly considering its association to the increased release of pro-inflammatory cytokines and to the reduction of zinc release and MTs synthesis involved in the control of the inflammatory response. These data strongly suggest that the genetic screening of the -308G/A polymorphism may be a valid tool for identification of subjects needing a more appropriate therapy when affected by acute and/or recurrent infectious diseases.

Abstract: Zn2+ is a potently toxic cation involved in the neuronal injury observed in cerebral ischemia, epilepsy, and brain trauma. Toxic Zn2+ accumulation may result from either trans-synaptic Zn2+movement and/or cation mobilization from intracellular sites. To gain entry to the cytosol, Zn2+ can flux through glutamate receptor-associated channels, voltage-sensitive calcium channels, or Zn2+-sensitive membrane transporters, while metallothioneins and mitochondria provide sites of intracellular Zn2+ release. Intracellular Zn2+ homeostasis is sensitive to patho-physiological environmental changes, such as acidosis, inflammation and oxidative stress. The mechanisms by which Zn2+ exerts its neurotoxicity include mitochondrial and extra-mitochondrial production of reactive oxygen species and disruption of metabolic enzymatic activity, ultimately leading to activation of apoptotic and/or necrotic processes. Beside acute neuronal injury, an exciting new area of investigation is offered by the role of Zn2+ dysmetabolism in Alzheimer's disease as the cation acts as a potent trigger for Abeta aggregation and plaque formation. Finally, recent findings suggest that alteration of Zn2+ homeostasis might also be a critical contributor to aging-related neurodegenerative processes. Thus, multiple evidence suggest that modulation of intracellular and extracellular Zn2+ might be an important therapeutical target for the treatment of a vast array of neurological conditions ranging from stroke to Alzheimer's disease.

Abstract: Vascular complications, including ischaemic cardiomyopathy, are the major causes of death in old diabetic patients. Chronic inflammation due to high IL-6 production occurs in type 2 diabetes (NIDDM) and atherosclerosis. High levels of IL-6 are associated with hyperglycaemia, dyslipidemia and provoke insulin resistance. In ageing and inflammation, IL-6 affects Metallothionein (MT) homeostasis, which in turn is involved in zinc turnover. Zinc deficiency is an usual event in ageing, inflammation, type 2 diabetes and atherosclerosis. No genetic study exists on MT polymorphisms in NIDDM-atherosclerotic patients. The aim of the present study is to screen a single nucleotide polymorphism in the promoter region of the MT2A gene in relation to inflammation (IL-6) and plasma zinc in NIDDM-atherosclerotic patients. The -209 A/G MT2A polymorphism is associated with chronic inflammation (higher plasma levels of IL-6), hyperglycaemia, enhanced HbA1c and more marked zinc deficiency in AA than AG genotype carrying patients. Analysing patients and controls subdivided in AA and AG genotypes, significant interactions existed between disease status and genotypes for glucose and zinc. AA patients are more at risk of developing NIDDM in association with atherosclerosis (p=0.0015 odds ratio=2.617) and its complications, such as ischaemic cardiomyopathy (p=0.0050 odds ratio=12.6). In conclusion, high levels of IL-6 unmask the phenotypes (higher insulin resistance and zinc deficiency) in relation to the genotypes with subsequent risk of developing ischaemic cardiomyopathy in NIDDM-atherosclerotic patients carrying AA genotype. Hence, the novel -209A/G MT2A polymorphism may be a further useful tool for the prevention, diagnosis and therapy of these combined pathologies in the elderly.

Abstract: Neutrophils are the first barrier against infections. Aged neutrophils display impaired oxidative burst and phagocytosis with subsequent less capability to destroy bacteria. In successful ageing (nonagenarians), neutrophil efficiency (phagocytosis) increases. After ingested microbes, aged neutrophils are less prone to undergo apoptosis favouring chronic inflammation. Moreover, the superoxide dismutase (SOD) activity, which is necessary in avoiding ROS produced by oxidative burst, is limited in ageing. The mechanisms of age-related changes in neutrophil function are not fully understood, taking also into account that nonagenarians escape infections in comparison with elderly. Zinc pool may be involved because it is pivotal for neutrophil efficiency and SOD activity. Since zinc also controls the inflammation, via IL-6 and soluble factor of gp130 (sgp130), we have assessed the possible interrelationship among oxidative burst, apoptosis, inflammation, SOD, adhesion molecule Mac-1 and zinc pool in elderly and in nonagenarians. The oxidative burst and the capacity to increase Mac-1 after PMA stimulation decrease both in elderly and nonagenarians, but the latter display a slight increased neutrophil induced apoptosis, decreased sgp130, increased SOD, and more neutrophil zinc content, as it occurs in young-adults. Significant correlation exists between sgp130 and zinc pool in very old age. These findings suggest lower chronic inflammation in nonagenarians, via more zinc available, with subsequent long-life survival. Therefore, a more correct interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool exists in successful ageing with subsequent more effectiveness to control the inflammatory response to pathogens.

Abstract: The capacity of the remodeling immune responses during stress (immune plasticity) is fundamental to reach successful aging. We herein report two pivotal models to demonstrate the relevance of the immune plasticity in aging and successful aging. One model is represented by the circadian rhythms of immune responses; the other one is the immune responses during partial hepatectomy/liver regeneration (pHx). The latter is suggestive because it mimics the immunosenescence and chronic inflammation 48 hours after partial hepatectomy in the young through the continuous production of IL-6, which is the main cause of immune plasticity lack in aging. The constant production of IL-6 leads to abnormal increments of zinc-bound metallothionein (MT), which is, in turn, unable in zinc release in aging. As a consequence, low zinc ion bioavailability appears for thymic and extrathymic immune efficiency, in particular, of liver NKT cells bearing TCR gd. The remodeling during the circadian cycle and during pHx of zinc-bound MT confers the immune plasticity of liver NKT gamma delta cells and NK cells in young and very old age, not in old age. Therefore, zinc-bound MT homeostasis is crucial in conferring liver immune plasticity with subsequent successful aging.

Abstract: NKT cells derive from the thymus and home to the liver. Liver NKT cells can be divided in two groups: 'classical' and 'non-classical'. The first is CD1d-restricted, the second is CD1d-unrestricted. NKT cells (classical and non-classical) co-express T-cell receptor (TCR) and NK-cell marker (NK1.1), display cytotoxicity and produce IFN-gamma under IL-12 stimulation affecting, thereby, Th1 response and innate immunity. NK1.1(+)TCR alpha/beta(+) cells belong to both groups. NK1.1(+)TCR gamma/delta(+) cells belong to the second group. Anyway, both NKT cell subtypes, via IFN-gamma production, protect against viruses and bacteria from early in life. Immune variations as well as zinc rhythmicity during the circadian cycle confer the immune plasticity, which is essential for successful ageing. Liver NK1.1(+)TCR gamma/delta(+) cells, rather than TCR alpha/beta(+), from young and very old mice display 'in vitro' (under IL-12 stimulation) nocturnal peaks in cytotoxicity and IFN-gamma production. The acrophase of liver NK1.1(+)TCR gamma/delta(+) cells is present in young and very old mice, not in old. The interplay among zinc-bound metallothionein (MT)/IL-6/gp130/poly(ADP-ribose) polymerase-1 (PARP-1) may be involved in conferring plasticity to liver NK1.1(+)TCR gamma/delta(+) cells. IL-6, via sub-unit receptor gp130, induces MTmRNA. At night, gene expressions of MT, IL-6, gp130 are lower in very old mice than old and young MT-I transgenic mice (MT-I*). In very old mice, this phenomenon allows limited sequester of intracellular zinc from MT leading to good free zinc ion bioavailability for immune efficiency and zinc-dependent PARP-1 activity. Indeed (1) in vitro, high IL-6 provokes strong accumulation of MT, impaired cytotoxicity and low zinc ion bioavailability in liver NK1.1(+)TCR gamma/delta(+) cells exclusively from old and MT-I* mice. (2) The ratio total/endogen PARP-1 activity is higher in very old than in old and MT-I* mice, suggesting a higher capacity of PARP-1 in base excision DNA-repair in very old age thanks to low zinc-bound MT. Cytotoxicity and IFN-gamma production from liver NK1.1(+)TCR gamma/delta(+) cells are thus preserved leading to successful ageing. In conclusion, MT/IL-6/gp130/PARP-1 interplay may confer plasticity to liver CD1d-unrestricted NK1.1(+)TCR gamma/delta(+) cells, where MT, IL-6, gp130 are the main upstream protagonists, and PARP-1 is the main downstream protagonist in immunosenescence.

Abstract: Metallothionein (MT)-III isoform is a brain metal-binding protein that, like the MT-I + II isoform, binds zinc with high affinity. In the young-adult age, MT-III isoform increases during transient stress while MT-I + II isoform decreases, suggesting compensatory phenomena between the two isoforms and a protective role of MT-III against oxidative damage. This role may be questioned during ageing, because the stress-like condition is chronic in ageing due to high persistent levels of interleukin-6. In the present study, high expression of MT-III and MT-I + II genes (examined by RT-PCR and in situ hybridisation) was found in the hippocampus of old rats. These results indicate that a large amount of free zinc ions can be sequestered by MT isoforms, leading to impaired zinc-dependent functions in the ageing brain. In addition, zinc (tested with the Timm's method) was found to be low in mossy fibres from the old hippocampus. As this method tests bound and unbound zinc, we also investigated free zinc ion bioavailability based on the ratio active thymulin/total thymulin. We found that zinc ion bioavailability was low in old rats, together with increased interleukin-6 mRNA, high expression of both MT isoforms and reduced number of synapses whose function is zinc-dependent, in the old hippocampus. The results indicate that concomitant increments of both MT isoforms may provoke detrimental synergistic effects leading to reduced free zinc ion bioavailability for synapses. As a consequence, compensatory phenomena between MT isoforms may not occur in the old hippocampus due to chronic stress-like condition elicited by high persistent levels of interleukin-6.

Abstract: Immunosenescence is defined as the state of dysregulated immune function that contributes to the increased susceptibility to infection, cancer and autoimmune diseases observed in old organisms, including humans. However, dysregulations in the immune functions are normally counterbalanced by continuous adaptation of the body to the deteriorations that occur over time. These adaptive changes are likely to occur in healthy human centenarians. Both innate (natural) and adaptive (acquired) immune responses decline with advancing age. Natural killer (NK) and natural killer T (NKT) cells represent the best model to describe innate and adaptive immune response in aging. NK and NKT cell cytotoxicity decreases in aging as well as interferon-gamma (IFN-gamma) production by both activated cell types. Their innate and acquired immune responses are preserved in very old age. However, NKT cells bearing T-cell receptor (TCR) gammadelta also display an increased cytotoxicity and IFN-gamma production in very old age. This fact suggests that NKT cells bearing TCRgammadelta are more involved in maintaining innate and adaptive immune response in aging leading to successful aging. The role played by the neuroendocrine-immune network and by nutritional factors, such as zinc, in maintaining NK and NKT cell functions in aging is discussed.

Abstract: BACKGROUND: With advancing age, thymic efficiency shows progressive decline due to thymic involution allowing impaired cell-mediated immunity and the appearance of age-related diseases. The intrinsic cause of thymic involution is still undefined. Chronic inflammation and high glucocorticoids (GCs) may be involved. However, transgenic mice, with increased GC sensitivity and over expression of GC receptors, display delayed age-associated thymic involution. This fact suggests that other substances may affect thymic involution. Among them, both isoforms of metallothioneins (MTs) I+II and III are the major candidates because their increments leads to organ atrophy in constant stress and are induced by IL-6, which increases in ageing. Enhanced MTs in ageing allows constant sequester of zinc ions and no subsequent zinc release leading to low zinc ion bioavailability for thymic efficiency. This sequester is very limited in very old age. Thus, we have investigated the MTmRNA (I+II and III) in the thymus from young, old and very old mice. METHODS: MTmRNA and IL-6mRNA (RT-PCR) in the thymus from different donors were tested. Concomitantly, TECs proliferation, zinc ion bioavailability (ratio total thymulin/active thymulin), thymulin activity and corticosterone were tested from different donors. RESULTS: Both isoforms of MTmRNA and IL-6mRNA increase in old thymus coupled with low zinc ion bioavailability, reduced TECs proliferation, impaired thymulin activity and enhanced plasma corticosterone in comparison with young. Conversely, although the thymus is involuted in very old mice because of no changes in thymus weight in comparison to old mice, reduced MTmRNA, especially MT-I+II isoforms, and low IL6mRNA occur. Concomitantly, good zinc ion bioavailability, maintained TECs proliferation, satisfactory thymulin activity and reduced corticosterone are observed in very old mice. CONCLUSIONS: The concomitant increments by high IL-6 of both MT isoforms in the thymus from old mice may be involved in thymic involution because provoking low zinc ion bioavailability, which is relevant for thymic efficiency. By contrast, the limited increments of MTs by low IL-6 induce good zinc ion bioavailability and satisfactory thymic efficiency in very old mice. Therefore, abnormal increased MTs may provoke complete thymic involution during ageing and the possible appearance of age-related diseases. If their increments are instead limited by low inflammation, healthy ageing and longevity may be reached.

Abstract: The use of HPLC coupled on-line with a mass spectrometer is a very powerful tool in order to analyze intact PLs molecular species (PMS) without the need of derivatization, thus decreasing the risk of artifacts formation. A normal-phase HPLC-ESI-MS-MS method has been developed in order to study the human blood mononuclear cell PMS composition. This method was applied to characterize PMS from seven CF subjects and from seven age-matched healthy subjects. More than 140 phospholipid molecular species from phosphatidylethanolamine (PE), plasmalogen phosphatidylethanolamine (pPE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC) and sphingomyelin (Sph) were identified and compared. Differences between the two groups were found in pPE (p16:0/22:6), pPE (p18:0/22:6), PE (16:0/20:4) and PC (16:0/18:2) which were significantly lower in CF subjects and in PC (16:0/16:1) which was significantly higher in CF subjects.

Abstract: Human centenarians attract increasing interest as they hold some still undefined molecular mechanisms resulting in the achievement of exceptional old age. Recent data suggest the ability of centenarians to efficiently counter the increased cellular stress normally associated with ageing. The ubiquitous heat shock (HS) protein HSP70, expressed under the control of the heat shock transcription factor 1 (HSF-1), is recognized as one of the main chaperones associated with cell protection against stresses. In fact, HSP70 protein induction by heat, a classic well characterized cellular stress, was recently reported to be reduced in cells of most aged humans but not in centenarians. In order to investigate the molecular basis of this feature, we analyzed in vitro the time course expression of the hsp70 gene and the activation of HSF-1 in heat treated Epstein Barr virus transformed B-lymphocytes of centenarians. Our study demonstrates that lymphoblasts from centenarians maintain the transcriptional response of hsp70 gene to heat stress similar to young subjects. Such normal induction of hsp70 is associated to higher binding activity of HSF-1 that compensates an age-dependent delay in HSF-1 phosphorylation. Moreover, in vitro zinc supplementation had an age-dependent effect on hsp70 expression, indicating a role for this nutritionally important molecule and suggesting its involvement in cellular stress responses.

Abstract: Since phosphatidylcholine (PC) is the most abundant phospholipid (PL) class in human serum, its concentration represents an important marker for the evaluation of lipid absorption and metabolism. High-performance liquid chromatography coupled on-line with electrospray ionization ion-trap tandem mass spectrometry (HPLC/ESI-MS/MS) was successfully applied to the quantitative analysis of PC molecular species from serum of cystic fibrosis (CF) subjects before and after supplementation with docosahexaenoic acid (DHA). Seven molecular species of PC (containing C16:0/C20:4, C16:0/C22:6, C18:0/C20:4, C18:0/C22:6, C16:0/C18:1, C16:0/C18:2 and C18:0/C18:2, respectively) were quantified using MS in the negative scan mode with 1,2-diundecanoyl-sn-glycero-phosphocholine as the internal standard. The molecular species containing DHA, C16:0/C22:6 and C18:0/C22:6, increased from 41.3 +/- 31.7 and 33.1 +/- 18.2 to 85.4 +/- 20.4 and 52.1 +/- 20.7 microg/mL serum, respectively, after a 3-month supplementation. Interestingly, the species containing arachidonic acid (C18:0/C20:4 and C16:0/C20:4) decreased from 115 +/- 55 and 139 +/- 57 to 58.1 +/- 22.5 and 70.5 +/- 28.1, respectively. HPLC/ESI-MS/MS allowed the direct analysis of the lipid extract without previous purification of PLs, thus it is a useful analytical support in CF research in order to understand the extent of lipid dysfunctions typical of CF or other diseases. The present method might also be used for quantitative analysis of each serum phospholipid class molecular species. However, the instrument response was found to be very dependent on the phospholipid class considered, and thus the use of appropriate standards for each class of PLs is recommended.

Abstract: BACKGROUND: The increasing demand for health care services, the extraordinary results obtained by applying innovative biotechnology, and the intense debate aroused by the possibility that postponement of aging may be in sight should promote public enthusiasm and funding for biomedical aging research aimed at enhancing a healthy life span by healthy productive life. OBJECTIVE: New trends in biomedical aging research arise from the advancement of knowledge on the basic molecular mechanisms of aging and the availability of advanced biotechnology. RESULTS: The emerging scenario is that, in opposition to the highly publicized, yet unwarranted claims of existing so-called anti-aging treatments, innovative and promising therapies for the treatment and prevention of age-related diseases and intriguing strategies aimed at the postponement of aging are currently in the process of development. CONCLUSIONS: Although many problems will remain unsolved for long, it seems that a common consensus has been raised towards the possible serious impact of these strategies on the development of knowledge around the mechanisms of aging.

Abstract: Cystic fibrosis (CF) is characterized by abnormal levels of essential fatty acids (EFA) in plasma phospholipids. The reduced availability of EFA has been reported to alter patterns of circulating and tissue esterified acids and may determine profound changes in membrane fluidity and cell signaling mechanisms. In the current study, the results of a new strategy aimed at the realization of a practical, low cost integrator, for daily use in the dietary management of FC subjects, are reported. We investigated the plasma phospholipids and triglycerides fatty acids composition of CF patients subjected to a dietary supplement constituted of a mixture of 50% extra virgin olive oil and 50% soybean oil and studied the clinical effects of this supplementation. The study included fourteen young subjects, aged between 6 and 15 years, affected by cystic fibrosis, with pancreatic insufficiency and heterozygotes or homozygotes for the delta F508 mutation. The subjects were matched by age and randomly assigned to either an oil mixture supplemented (OM) group (n = 7), or to a control (C) group (n = 7). In contrast to the control group, the patients with supplemented diet achieved significant increases of the relative amount of C18:1 in the triglycerides as well as a significant decrease in saturated fatty acids (C 16:0, C 17:0, C 18:0, C 22:0). Moreover, the ratio between LA acid and AA significantly increased in the triglycerides of the OM group. In the phospholipids of the OM group, the relative amount of C 18:1 and of palmitic acid increased significantly whereas the relative amount of the most important polyunsaturated fatty acids (PUFA) decreased. These results show that oleic acid can be absorbed and incorporated into the plasma triglycerides of CF patients receiving pancreatic enzymes, whereas poor incorporation of LA occurs. Despite the reduction in the relative amounts of phospholipid PUFA, the supplemented subjects did not reported adverse effects There were no significant differences between groups in the clinical indexes recorded (height, weight, BMI, Schwachman-Kulczycki score and FEV 1s). The results of this study showed that the supplementation with a mixture of extravirgin olive and soybean oil was safe in seven CF patients treated during a 2-months period and no negative clinical effects were evident. However, further clinical trials will be necessary in order to better evaluate the consequence of the observed changes in plasma fatty acids composition in a longer testing period.

Abstract: An ever increasing number of people have been engaging in aging research using various interventions aimed to modify aging processes, and/or life span, of experimental animals. Since this type of studies needs outcome parameters for assessing the efficacy of such interventions, research on biomarkers of aging (ABs) has received new stimuli. In the present paper, the problem of the occurrence of a vicious circle any time we study ABs and determinants of aging is addressed. In fact, while ABs would represent the standard reference to be used in the study of the main causes of processes of aging, these very determinants should already be known in order to get reliable ABs. A feasible way to overcome this impasse is proposed, using mathematical models of survivorship or mortality based on biological hypotheses and accounting for inter-individual heterogeneity, a necessary ingredient for a correct interpretation of survival results. Specific kinetics of experimental parameters that are candidates as ABs can be compared to the kinetics hypothesized for general biological functions entering the model. We have built a model of this type that can also be used to perform a reliable overall gross estimate of the rate of aging, R(a), in the population, a parameter useful when judging the success of interventions aimed to act on determinants of aging. The perspective that theory of complex systems can be of help in the search for ABs is also discussed.

Abstract: The Lewis acid-mediated reduction of alpha-alkyl-beta-keto esters has been shown to proceed by different stereochemical control depending on the nature of the metal atom. Strongly chelating TiCl(4) led to the syn isomer in high diastereomeric excess in noncoordinating solvents (CH(2)Cl(2)) at -78 degrees C with BH(3).py as reducing agent, while nonchelating CeCl(3) gave a high excess of the anti isomer in coordinating solvents (THF) at the same temperature with lithium triethylborohydride (LiEt(3)BH) as reducing agent. The methodology has been successfully utilized for obtaining important syn- and anti-alpha-alkyl-beta-hydroxy esters with high diastereoselectivity.