Zinco, invecchiamento e sistema immunitario: effetti sull'apoptosi e sulla proliferazione dei linfociti

Immunosenescence is characterized by a complex remodelling of the immune system, mainly driven by lifelong antigenic burden. Cells of the immune system are constantly exposed to a variety of stressors capable of inducing apoptosis, including antigens and reactive oxygen species continuously produced during immune response and metabolic pathways. The overall homeostasis of the immune system is based on the balance between antigenic load, oxidative stress, and apoptotic processes on one side, and the regenerative potential and renewal of the immune system on the other. Zinc is an essential trace element playing a central role on the immune function, being involved in many cellular processes, such as cell death and proliferation, as cofactor of enzymes, nuclear factors and hormones. In this context, the age associated changes in the immune system may be in part due to zinc deficiency, often observed in aged subjects and able to induce impairment of several immune functions. Thus, the aim of this work was to investigate the role of zinc in two essential events for immunity during aging, i.e. apoptosis and cell proliferation. Spontaneous and oxidative stress-induced apoptosis were evaluated by flow cytometry in presence of a physiological concentration of zinc in vitro on peripheral blood mononuclear cells (PBMCs) obtained from healthy subjects of different age: a group of young subjects, a group of old subjects and a group of nonagenarians. In addition, cell cycle phases were analyzed by flow cytometry in PBMCs, obtained from the subjects of the same groups in presence of different concentration of zinc. We also analyzed the influence of zinc in these processes in relation to p53 codon 72 polymorphism, known to affect apoptosis and cell cycle in age-dependent manner. Zinc significantly reduces spontaneous apoptosis in all age-groups; while it significantly increases oxidative stress-induced late apoptosis/necrosis in old and nonagenarians subjects. Some factors involved in the apoptotic pathway were studied and a zinc effect on mitochondrial membrane depolarization, cytochrome C release, caspase-3 activation, PARP cleavage and Bcl-2 expression was found. In conclusion, zinc inhibits spontaneous apoptosis in PBMCs contrasting the harmful effects due to the cellular culture conditions. On the other hand, zinc is able to increase toxicity and induce cell death in PBMCs from aged subjects when cells are exposed to stressing agents that compromise antioxidant cellular systems. Concerning the relationship between the susceptibility to apoptosis and p53 codon 72 genotype, zinc seems to affect apoptosis only in PBMCs from Pro- people suggesting a role of this ion in strengthening the mechanism responsible of the higher propensity of Pro- towards apoptosis. Regarding cell cycle, high doses of zinc could have a role in the progression of cells from G1 to S phase and from S to G2/M phase. These effect seems depend on the age of the donor but seems to be unrelated to p53 codon 72 genotype. In order to investigate the effect of an in vivo zinc supplementation on apoptosis and cell cycle, PBMCs from a group of aged subjects were studied before and after six weeks of oral zinc supplementation. Zinc supplementation reduces spontaneous apoptosis and it strongly reduces oxidative stress-induced apoptosis. On the contrary, no effect of zinc was observed on cell cycle. Therefore, it’s clear that in vitro and in vivo zinc supplementation have different effects on apoptosis and cell cycle in PBMCs from aged subjects. Further experiments and clinical trials are necessary to clarify the real effect of an in vivo zinc supplementation because this preliminary data could encourage the of this element in all that disease with oxidative stress pathogenesis. Moreover, the expression of metallothioneins (MTs), proteins well known for their zinc-binding ability and involved in many cellular processes, i.e. apoptosis, metal ions detoxification, oxidative stress, differentiation, was evaluated in total lymphocytes, in CD4+ and in CD8+ T lymphocytes from young and old healthy subjects in presence of different concentration of zinc in vitro. Literature data reported that during ageing the levels of these proteins increase and concomitantly they lose the ability to release zinc. This fact induce a down-regulation of many biological functions related to zinc, such as metabolism, gene expression and signal transduction. Therefore, these proteins may turn from protective in young-adult age to harmful agents for the immune function in ageing following the concept that several genes/proteins that increase fitness early in life may have negative effects later in life: named “Antagonistic Pleyotropy Theory of Ageing”. Data obtained in this work indicate an higher and faster expression of MTs with lower doses of zinc in total lymphocytes, in CD4+ and in CD8+ T lymphocytes from old subjects supporting the antagonistic pleiotropic role of these proteins.

The relationship of thymulin activity, immune parameters of HIV disease progression and substance use in a cohort of HIV seropositive alcohol and drug users

Zinc is essential for the activity of thymulin, a thymic hormone involved in T-lymphocyte differentiation and activation. Zinc deficiency is widespread in populations with HIV infection, and HIV+ drug users are particularly susceptible to zinc deficiency and immune suppression. This dissertation explored the relationship of zinc-bound active thymulin to plasma zinc, CD4+ and CD8+ cell count, the CD4+/CD8+ ratio, and drug use in HIV-infected drug users. Zinc-bound active thymulin was assessed in plasma of HIV+ drug users who were participating in a 30 month zinc supplementation trial. Plasma from 80 participants at the 12 month visit, and 40 of these same participants, randomly selected, at the baseline visit were assessed for zinc-bound active thymulin levels using a modification of the rosette inhibition assay. Thymulin activity was directly associated with CD4+ cell count (β = 0.127, p = 0.002) and inversely associated with cocaine use (β = −0.908, p = 0.026; R2 = 0.188, p = 0.019) independent of HIV viral load, age, gender and antiretroviral use. An increase in thymulin activity was 1.4 times more likely when CD4+ cell count increased (OR = 1.402, 95%CI: 1.006–1.956), independent of change in viral load, antiretroviral use, and age. Participants who used cocaine consistently, were 7.6 times less likely to have an increase in thymulin activity (OR = 0.133, 95%CI: 0.017–1.061). There was a direct correlation between change in plasma zinc and change in zinc-bound active thymulin (r = 0.243, p = 0.13). Analysis of CD4+ cell count decline in 222 participants in the zinc supplementation trial across the 30 months showed that both crack cocaine use and heavy alcohol use accelerated CD4+ cell count decline. Thymulin activity is directly associated with HIV disease progression, measured by CD4+ cell count, and is depressed with cocaine use independent of antiretroviral use and HIV viral load. Cocaine and heavy alcohol accelerate CD4+ cell count decline. The effect of cocaine on thymic output requires further evaluation as a mechanism for the association of cocaine use with faster HIV disease progression.

Métodos de avaliação nutricional para a suplementação oral com zinco em crianças pré-púberes não deficientes em zinco

Background: Zinc is an essential nutrient that is required for numerous metabolic functions, and zinc deficiency results in growth retardation, cellmediated immune dysfunction, and cognitive impairment. Objective: This study evaluated nutritional assessment methods for zinc supplementation in prepubertal nonzinc- deficient children. Design: We performed a randomised, controlled, triple-blind study. The children were divided into a control group (10% sorbitol, n = 31) and an experimental group (10 mg Zn/day, n = 31) for 3 months. Anthropometric and dietary assessments as well as bioelectrical measurements were performed in all children. Results: Our study showed (1) an increased body mass index for age and an increased phase angle in the experimental group; (2) a positive correlation between nutritional assessment parameters in both groups; (3) increased soft tissue, and mainly fat-free mass, in the body composition of the experimental group, as determined using bioelectrical impedance vector analysis; (4) increased consumption of all nutrients, including zinc, in the experimental group; and (5) an increased serum zinc concentration in both groups (p < 0.0001). Conclusions: Given that a reference for body composition analysis does not exist for intervention studies, longitudinal studies are needed to investigate vector migration during zinc supplementation. These results reinforce the importance of employing multiple techniques to assess the nutritional status of populations.

Métodos de avaliação nutricional para a suplementação oral com zinco em crianças pré-púberes não deficientes em zinco

Background: Zinc is an essential nutrient that is required for numerous metabolic functions, and zinc deficiency results in growth retardation, cellmediated immune dysfunction, and cognitive impairment. Objective: This study evaluated nutritional assessment methods for zinc supplementation in prepubertal nonzinc- deficient children. Design: We performed a randomised, controlled, triple-blind study. The children were divided into a control group (10% sorbitol, n = 31) and an experimental group (10 mg Zn/day, n = 31) for 3 months. Anthropometric and dietary assessments as well as bioelectrical measurements were performed in all children. Results: Our study showed (1) an increased body mass index for age and an increased phase angle in the experimental group; (2) a positive correlation between nutritional assessment parameters in both groups; (3) increased soft tissue, and mainly fat-free mass, in the body composition of the experimental group, as determined using bioelectrical impedance vector analysis; (4) increased consumption of all nutrients, including zinc, in the experimental group; and (5) an increased serum zinc concentration in both groups (p < 0.0001). Conclusions: Given that a reference for body composition analysis does not exist for intervention studies, longitudinal studies are needed to investigate vector migration during zinc supplementation. These results reinforce the importance of employing multiple techniques to assess the nutritional status of populations.

Influência do zinco suplementar na maturação biológica e no crescimento de jovens atletas de futebol do sexo masculino

Introdução O zinco é um importante elemento traço que auxilia na capacidade antioxidante, além de participar da maturação biológica. Em atletas, a suplementação de zinco tem efeito positivo nos parâmetros hematológicos e pode melhorar o rendimento esportivo. Sua deficiência é comumente observada nesses grupo e pode estar associada à diminuição da força física assim como da massa corporal, além de ter efeito significativo no crescimento. Assim os objetivos do presente estudo foram: comparar os métodos de avaliação da maturidade biológica e suas relações com variáveis antropométricas e de rendimento físico de acordo com o estado de zinco em jovens jogadores de futebol; comparar diferentes métodos de avaliação da composição corporal em jovens jogadores de futebol esratificados de acordo com os níveis plasmáticos de zinco e investigar o efeito do zinco suplementar na maturação biológica, no crescimento, na composição corporal e na força muscular de jogadores de futebol púberes do sexo masculino. Materiais e métodos Foram avaliados em dois momentos 48 jovens do sexo masculino (13±1 anos, massa corporal de 48±10kg, estatura de 160±10cm e zinco plasmático de 12,1±2,2 μmol/L). Todos eram jogadores de futebol de um tradicional clube do Rio de Janeiro e foram divididos aleatoriamente em dois grupos. Durante 12 semanas o grupo placebo (n=28) recebeu cápsulas de amido de milho e o grupo suplementado (n=20) recebeu cápsulas de gluconato de Zn (22mg/dia). O valor de 11,0 μmol/L foi considerado como ponto de corte para classificação dos jovens em normozincêmicos ou hipozincêmicos. No início da manhã, após jejum noturno, foram coletados sangue e urina para determinação da concentração de zinco. A massa corporal, alturas (do vértex, acromial, dactiloidal, iliocristal, trocantérica e sentado), composição corporal, força e maturidade esquelética (TW3) também foram determinadas por metodologias validadas. Resultados As comparações entre as categorias maturacionais definidas por cada método de avaliação mostraram que a idade óssea foi o único método que permitiu a identificação de diferenças entre as três categorias, em ao menos duas variáeis relacionadas ao rendimento (massa livre de gordura (MLG) e força na mão dominante (FMD) – p<0,0001). O método da pilosidade axilar foi capaz de discriminar apenas para a FMD (p<0,0001). Embora tenha fornecido quatro categorias maturacionais, o método por dosagem da testosterona não possibilitou a identificação de diferenças entre as categorias relativas a MLG, a FMD e as dobras cutâneas(DC). Quando observamos os métodos de avaliação da composição corporal não foram identificadas diferenças sigificativas entre os grupos hipozincêmico e normozincêmico no percentagem de gordura(PG) nem na MLG obtidas através dos métodos da absortometria de dupla energia (DXA) (p=0,06076 e p=0,5638 respectivamente), das DC (p=0,6840 and 0,5087) e através da bioimpedância elétrica (BIA) (p=0,3475 and p=0,3475). Entre os diferentes métodos também não foi encontrada diferenças significativas (PG: p=0,1272 e p=0,3231 - MLG: p=0,9229 and p=0,8933 para os grupos hipozincêmico e normozincêmico, respectivamente). As correlações entre os métodos foram significativas (PG: r= 0,3414 a 0,9765 e p<0,0001 a 0,0133 - MLG: r=0,9533 a 0,9998 e p<0,0001). Fortes coeficientes de determinação foram obtidos nas regressões múltiplas dos valores do DXA com a equação de Slaughter na estimativa da PG (r=0,86; r2=0,928 e SEE=2,37%) e ainda maiores para MLG (r=0,98; r2=0,990 e SEE=1,18kg). Valores menores foram encontrados para as outras equações com DC e para BIA. Ao analisar os efeitos da suplementação de zinco sobre o crescimento, a maturação, a composição corporal e a força, observou-se que somente as alterações ocorridas nos indicadores de crescimento foram significativas (p=0,0312), sendo que todas as demais não foram significativas - idade óssea (p=0,1391), massa livre de gordura (p=0,0593), percentual de massa gorda (p=0,2212) e força na mão dominante (p=0,6569). Conclusões Observando diferentes métodos de avaliação da maturidade biológica e as categorias por eles definidas, o método da idade óssea (IO) mostrou ser melhor, visto que ele permitiu identificar diferenças entre as três categorias possíveis, nas variáveis MLG e FMD, ao contrário dos outros métodos. Para a avaliação da composição corporal, os métodos baseados nas DC foram melhores que BIA, quando DXA não estiver disponível. A comparação entre os métodos baseados nas DC mostrou que a melhor associação foi obtida com a equação de Slaughter, seguida pela equação de Lohman com a utilização da IO ao invés da idade cronológica. Os níveis de zinco plasmático parecem não serem influenciados pela composição corporal, o que certamente justifica mais estudos. Os resultados da análise dos efeitos da suplementação de zinco no crescimento, na maturação, na composição corporal e na força, nos levam a concluir que o crescimento teve alteração positiva significativa e que os valores das demais variáveis estudadas (maturação, composição corporal e força muscular) não sofreram alterações significativas relacionadas à suplementação de zinco nos jovens jogadores de futebol do sexo masculino, na faixa etária dos 12 aos 14 anos.

Effect of zinc and manganese supplementation to tricalcium phosphate rich diet for tiger puffer (Takifugu rubripes)

Effects of zinc (Zn) and manganese (Mn) supplementation to a tricalcium phosphate (TCP) rich diet for tiger puffer have been investigated. A TCP supplement to the diet decreased the growth of fish compared to the control diet with a Ca supplementation from Ca-lactate. However, addition of either Zn or Mn to the TCP supplemented diet could not improve the growth of tiger puffer. Addition of both zinc and manganese to the TCP supplemented diet improved the growth of tiger puffer.

Effects of Cigarette Smoke and Ethanol Intake on Mouse Oesophageal Mucosa Changes Induced by Dietary Zinc Deficiency and Deoxycholic Acid Supplementation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Supplementation of diets for Santa Ines sheep with organic and inorganic zinc sources

This research was conducted with objective to evaluate the effect of different zinc (Zn) sources and doses in the diet for Santa Ines sheep. Forty lambs at weaning, with 18.4 kg of body weight were supplemented with three different sources of zinc (zinc oxide (ZnO), zinc amino acid and zinc proteinate) and three doses of zinc (200, 400 and 600 mg/kg DM) added to the basal diet. At every 28 days, animals were weighted and blood samples were collected for analyses of zinc (Zn), alkaline phosphatase and immunoglobulin G (IgG) and M (IgM). At the end of experiment, liver samples were collected for determination of the hepatic zinc levels. Zinc was analyzed with atomic absorption spectrophotometer, while phosphatase alkaline and immunoglobulins G and M were analyzed using Laborlab and Bioclin kits, respectively. There was no effect of diets on phosphatase alkaline levels and hepatic zinc, but there was difference in the plasmatic zinc levels and IgG and IgM levels. Based on the accumulation of hepatic zinc, the estimate of the zinc bioavailability, through the regression equation, showed that supplementation with organic and inorganic sources of zinc did not differ in the diet of Santa Ines sheep. © 2012 Sociedade Brasileira de Zootecnia.

Supplementation of diets for Santa Ines sheep with organic and inorganic zinc sources

This research was conducted with objective to evaluate the effect of different zinc (Zn) sources and doses in the diet for Santa Ines sheep. Forty lambs at weaning, with 18.4 kg of body weight were supplemented with three different sources of zinc (zinc oxide (ZnO), zinc amino acid and zinc proteinate) and three doses of zinc (200, 400 and 600 mg/kg DM) added to the basal diet. At every 28 days, animals were weighted and blood samples were collected for analyses of zinc (Zn), alkaline phosphatase and immunoglobulin G (IgG) and M (IgM). At the end of experiment, liver samples were collected for determination of the hepatic zinc levels. Zinc was analyzed with atomic absorption spectrophotometer, while phosphatase alkaline and immunoglobulins G and M were analyzed using Laborlab and Bioclin kits, respectively. There was no effect of diets on phosphatase alkaline levels and hepatic zinc, but there was difference in the plasmatic zinc levels and IgG and IgM levels. Based on the accumulation of hepatic zinc, the estimate of the zinc bioavailability, through the regression equation, showed that supplementation with organic and inorganic sources of zinc did not differ in the diet of Santa Ines sheep.

Effects of dietary phosphorus and zinc levels on growth artd bone mineralization in fingerlings of rainbow trout, Oncorhynchus mykiss

A laboratory based 2 x 2 factorial experiment was conducted to investigate the influences of dietary phosphorus and zinc levels on growth and bone mineralization in fingerlings of rainbow trout for 21 weeks. Two levels of phosphorus (19 and 30 mg/g) and two levels of zinc (55 and 103 Ag/g) in the dry diets were tested. Duplicate tanks of 30 rainbow trout (average weight 1.56 ± 0.24 g) per 60L glass tank were fed experimental diets three times a day to apparent satiation level at 15 to 24°C water temperature. The results of the present study demonstrated that dietary phosphorus supplementation influenced the growth and bone mineralization whereas zinc levels significantly (p<0.05) influenced bone mineralization in rainbow trout. Further investigations in this area with different size and age groups of this fish are broadly needed.

Green tea supplementation increases glutathione and plasma antioxidant capacity in adults with the metabolic syndrome

Green tea, a popular polyphenol-containing beverage, has been shown to alleviate clinical features of the metabolic syndrome. However, its effects in endogenous antioxidant biomarkers are not clearly understood. Thus, we tested the hypothesis that green tea supplementation will upregulate antioxidant parameters (enzymatic and nonenzymatic) in adults with the metabolic syndrome. Thirty-five obese participants with the metabolic syndrome were randomly assigned to receive one of the following for 8 weeks: green tea (4 cups per day), control (4 cups water per day), or green tea extract (2 capsules and 4 cups water per day). Blood samples and dietary information were collected at baseline (0 week) and 8 weeks of the study. Circulating carotenoids (a-carotene, ß-carotene, lycopene) and tocopherols (a-tocopherol, ?-tocopherol) and trace elements were measured using high-performance liquid chromatography and inductively coupled plasma mass spectroscopy, respectively. Serum antioxidant enzymes (glutathione peroxidase, glutathione, catalase) and plasma antioxidant capacity were measured spectrophotometrically. Green tea beverage and green tea extract significantly increased plasma antioxidant capacity (1.5 to 2.3 µmol/L and 1.2 to 2.5 µmol/L, respectively; P <.05) and whole blood glutathione (1783 to 2395 µg/g hemoglobin and 1905 to 2751 µg/g hemoglobin, respectively; P <.05) vs controls at 8 weeks. No effects were noted in serum levels of carotenoids and tocopherols and glutathione peroxidase and catalase activities. Green tea extract significantly reduced plasma iron vs baseline (128 to 92 µg/dL, P <.02), whereas copper, zinc, and selenium were not affected. These results support the hypothesis that green tea may provide antioxidant protection in the metabolic syndrome.