Zinc Abstracts 1

©

Copper excess, zinc deficiency, and cognition loss in Alzheimer's disease
            (Brewer, 2012) Download
In this special issue about biofactors causing cognitive impairment, we present evidence for and discuss two such biofactors. One is excess copper, causing neuronal toxicity. The other is zinc deficiency, causing neuronal damage. We present evidence that Alzheimer's disease (AD) has become an epidemic in developed, but not undeveloped, countries and that the epidemic is a new disease phenomenon, beginning in the early 1900s and exploding in the last 50 years. This leads to the conclusion that something in the developed environment is a major risk factor for AD. We hypothesize that the factor is inorganic copper, leached from the copper plumbing, the use of which coincides with the AD epidemic. We present a web of evidence supporting this hypothesis. Regarding zinc, we have shown that patients with AD are zinc deficient when compared with age-matched controls. Zinc has critical functions in the brain, and lack of zinc can cause neuronal death. A nonblinded study about 20 years ago showed considerable improvement in AD with zinc therapy, and a mouse AD model study also showed significant cognitive benefit from zinc supplementation. In a small blinded study we carried out, post hoc analysis revealed that 6 months of zinc therapy resulted in significant benefit relative to placebo controls in two cognitive measuring systems. These two factors may be linked in that zinc therapy significantly reduced free copper levels. Thus, zinc may act by lowering copper toxicity or by direct benefit on neuronal health, or both.

Zinc and glycemic control: a meta-analysis of randomised placebo controlled supplementation trials in humans
            (Capdor et al., 2013) Download
BACKGROUND: Impaired zinc metabolism is prominent in chronic disorders including cardiovascular disease and diabetes. Zinc has the potential to affect glucose homeostasis in animals and humans and hence impact the risk of type 2 diabetes mellitus. METHODS: A systematic review and meta-analysis of randomised placebo controlled trials was conducted to determine the effect of zinc supplementation on fasting blood glucose, HbA1c, serum insulin and serum zinc concentrations. Relevant studies for inclusion were identified from a literature search of electronic databases up to July 2011. RESULTS: Fourteen reports (n=3978 subjects) were included in the meta-analysis. In the overall analysis, a small but statistically significant reduction in fasting glucose concentrations was observed (-0.19+/-0.08mmol/L, P=0.013) after zinc supplementation. HbA1c tended to decrease in zinc-supplemented individuals (-0.64+/-0.36%, P=0.072). No significant effect was observed for serum insulin concentrations. Plasma zinc concentrations increased significantly following supplementation (+4.03+/-0.81mumol/L, P=0.001). In secondary analyses of participants with chronic metabolic disease (types 1 and 2 diabetes mellitus, metabolic syndrome and obesity), zinc supplementation produced a greater reduction in glucose concentrations (-0.49+/-0.11mmol/L, P=0.001) compared to the effect that was observed in healthy participants. CONCLUSION: The significant albeit modest reduction in glucose concentrations and tendency for a decrease in HbA1c following zinc supplementation suggest that zinc may contribute to the management of hyperglycemia in individuals with chronic metabolic disease.

Zinc and human health: an update
            (Chasapis et al., 2012) Download
The importance of micronutrients in health and nutrition is undisputable, and among them, zinc is an essential element whose significance to health is increasingly appreciated and whose deficiency may play an important role in the appearance of diseases. Zinc is one of the most important trace elements in the organism, with three major biological roles, as catalyst, structural, and regulatory ion. Zinc-binding motifs are found in many proteins encoded by the human genome physiologically, and free zinc is mainly regulated at the single-cell level. Zinc has critical effect in homeostasis, in immune function, in oxidative stress, in apoptosis, and in aging, and significant disorders of great public health interest are associated with zinc deficiency. In many chronic diseases, including atherosclerosis, several malignancies, neurological disorders, autoimmune diseases, aging, age-related degenerative diseases, and Wilson's disease, the concurrent zinc deficiency may complicate the clinical features, affect adversely immunological status, increase oxidative stress, and lead to the generation of inflammatory cytokines. In these diseases, oxidative stress and chronic inflammation may play important causative roles. It is therefore important that status of zinc is assessed in any case and zinc deficiency is corrected, since the unique properties of zinc may have significant therapeutic benefits in these diseases. In the present paper, we review the zinc as a multipurpose trace element, its biological role in homeostasis, proliferation and apoptosis and its role in immunity and in chronic diseases, such as cancer, diabetes, depression, Wilson's disease, Alzheimer's disease, and other age-related diseases.

Chemopreventive potential of zinc in experimentally induced colon carcinogenesis
            (Dani et al., 2007) Download
The present study was performed to evaluate the efficacy of zinc treatment on colonic antioxidant defense system and histoarchitecture in 1,2-dimethylhydrazine- (DMH) induced colon carcinogenesis in male Sprague-Dawley rats. The rats were segregated into four groups viz., normal control, DMH treated, zinc treated, DMH+zinc treated. Colon carcinogenesis was induced through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 16 weeks. Zinc (in the form of zinc sulphate) was supplemented to rats at a dose level of 227 mg/L in drinking water, ad libitum for the entire duration of the study. Increased tumor incidence, tumor size and number of aberrant crypt foci (ACF) were accompanied by a decrease in lipid peroxidation, glutathione-S-transferase, superoxide dismutase (SOD) and catalase. On the contrary, significantly increased levels of reduced glutathione (GSH) and glutathione reductase (GR) were observed in DMH treated rats. Administration of zinc to DMH treated rats significantly decreased the tumor incidence, tumor size and aberrant crypt foci number with simultaneous enhancement of lipid peroxidation, SOD, catalase and glutathione-S-transferase. Further, the levels of GSH and GR were also decreased following zinc supplementation to DMH treated rats. Well-differentiated signs of dysplasia were evident in colonic tissue sections by DMH administration alone. However, zinc treatment to DMH treated rats greatly restored normalcy in the colonic histoarchitecture, with no apparent signs of neoplasia. EDXRF studies revealed a significant decrease in tissue concentrations of zinc in the colon following DMH treatment, which upon zinc supplementation were recovered to near normal levels. In conclusion, the results of this study suggest that zinc has a positive beneficial effect against chemically induced colonic preneoplastic progression in rats induced by DMH.

Antioxidant vitamin and mineral supplements for slowing the progression of age-related macular degeneration
            (Evans and Lawrenson, 2012) Download
BACKGROUND: It has been proposed that antioxidants may prevent cellular damage in the retina by reacting with free radicals that are produced in the process of light absorption. Higher dietary levels of antioxidant vitamins and minerals may reduce the risk of progression of age-related macular degeneration (AMD). OBJECTIVES: The objective of this review was to assess the effects of antioxidant vitamin or mineral supplementation on the progression of AMD in people with AMD. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2012, Issue 8), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to August 2012), EMBASE (January 1980 to August 2012), Allied and Complementary Medicine Database (AMED) (January 1985 to August 2012), OpenGrey (System for Information on Grey Literature in Europe) (www.opengrey.eu/), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 20 August 2012. We searched the reference lists of identified reports and the Science Citation Index. We contacted investigators and experts in the field for details of unpublished studies. We also searched for systematic reviews of harms of vitamin supplements. SELECTION CRITERIA: We included randomised trials comparing antioxidant vitamin or mineral supplementation (alone or in combination) to placebo or no intervention in people with AMD. DATA COLLECTION AND ANALYSIS: Two authors assessed risk of bias and extracted data from the included trials. Where appropriate, we pooled data using a random-effects model unless three or fewer trials were available in which case we used a fixed-effect model. MAIN RESULTS: Thirteen trials (6150 participants) were included in this review. Over half the participants (3640) were randomised in one trial (AREDS in the USA), which found a beneficial effect of antioxidant (beta-carotene, vitamin C and vitamin E) and zinc supplementation on progression to advanced AMD (adjusted odds ratio (OR) 0.68, 95% confidence interval (CI) 0.53 to 0.87) over an average of 6.3 years. People taking supplements were less likely to lose 15 or more letters of visual acuity (adjusted OR 0.77, 95% CI 0.62 to 0.96). The other trials, in general, had shorter follow-up (less than two years). No evidence for an effect of supplementation was seen in these smaller trials of shorter duration. Overall we considered the strength of the evidence to be moderate. We did not consider included trials, in general, to be at risk of bias, although we found it difficult to assess reporting biases. The main reason for downgrading the strength of the evidence was because, for several analyses, only one trial was included and therefore consistency of the findings could not be assessed. The included trials reported the following adverse effects: hospitalisation for genito-urinary problems was more common in people taking zinc and yellowing of skin was more common in people taking antioxidants. Systematic searching of the literature identified other potential harms of vitamin supplementation, in particular an increased risk of lung cancer in smokers associated with beta-carotene supplements, but we were unable to identify a good systematic review of the evidence for harms of nutritional supplementation. AUTHORS' CONCLUSIONS: People with AMD may experience delay in progression of the disease with antioxidant vitamin and mineral supplementation. This finding is drawn from one large trial conducted in a relatively well-nourished American population. The generalisability of these findings to other populations is not known. Although generally regarded as safe, vitamin supplements may have harmful effects. A systematic review of the evidence on harms of vitamin supplements is needed.

Zinc and the risk for infectious disease
            (Fischer Walker and Black, 2004) Download
Zinc is an essential micronutrient for human growth, development, and immune function. Zinc deficiency impairs overall immune function and resistance to infection. Mild to moderate zinc deficiency can be best detected through a positive response to supplementation trials. Zinc supplementation has been shown to have a positive effect on the incidence of diarrhea (18% reduction, 95% CI: 7-28%) and pneumonia (41% reduction, 95% CI: 17-59%), and might lead to a decrease in the incidence of malaria. Zinc has also proven to decrease the duration of diarrhea by 15% (95% CI: 5-24%). Maternal zinc supplementation may lead to a decrease in infant infections. Studies assessing the role of zinc supplementation among persons with HIV, tuberculosis, and the common cold have not been conclusive. Two studies have shown zinc supplementation to decrease child mortality by more than 50%. Zinc clearly has an important role in infant and childhood infectious diseases; programs to increase the intake of zinc among deficient populations are needed.

Correlation between zinc status and immune function in the elderly
            (Haase et al., 2006) Download
Zinc is essential for the immune system and elderly people have an increased probability for zinc deficiency, documented by a decline of serum or plasma zinc levels with age. Although most healthy elderly are not classified as clinically zinc deficient, even marginal zinc deprivation can affect immune function. Several striking similarities in the immunological changes during aging and zinc deficiency, including a reduction in the activity of the thymus and thymic hormones, a shift of the T helper cell balance towards TH2, decreased response to vaccination, and impaired functions of innae immune cells indicate that a wide prevalence of marginal zinc deficiency in elderly people may contribute to immunosenescence. Studies with oral zinc supplementation show the potential to improve the immune response of elderly people by restoration of the zinc levels, showing that balancing the zinc status may be a way to healthy aging. This review summarizes the current literature about zinc supplementation in the elderly and thereby defines the rationale for the immunological part of the ZINCAGE project.

Effects of zinc supplementation on diabetes mellitus: a systematic review and meta-analysis
            (Jayawardena et al., 2012) Download
The number of people with diabetes and pre-diabetes are exponentially increasing. Studies on humans have shown the beneficial effects of Zinc supplementation in patients with diabetes. The present study aims to systematically evaluate the literature and meta-analyze the effects of Zinc supplementation on diabetes. A systematic review of published studies reporting the effects of Zinc supplementations on diabetes mellitus was undertaken. The literature search was conducted in the following databases; PubMed, Web of Science and SciVerse Scopus. A meta-analysis of studies examining the effects of Zinc supplementation on clinical and biochemical parameters in patients with diabetes was performed. The total number of articles included in the present review is 25, which included 3 studies on type-1 diabetes and 22 studies on type-2 diabetes. There were 12 studies comparing the effects of Zinc supplementation on fasting blood glucose in patients with type-2 diabetes. The pooled mean difference in fasting blood glucose between Zinc supplemented and placebo groups was 18.13mg/dl (95%CI:33.85,2.41; p<0.05). 2-h post-prandial blood sugar also shows a similar distinct reduction in (34.87mg/dl [95%CI:75.44; 5.69]) the Zinc treated group. The reduction in HbA1c was 0.54% (95%CI:0.86;0.21) in the Zinc treated group. There were 8 studies comparing the effects of Zinc supplementation on lipid parameters in patients with type-2 diabetes. The pooled mean difference for total cholesterol between Zinc supplemented and placebo groups was 32.37mg/dl (95%CI:57.39,7.35; p<0.05). Low-density lipoprotein cholesterol also showed a similar distinct reduction in the Zinc treated group, the pooled mean difference from random effects analysis was 11.19mg/dl (95%CI:21.14,1.25; p<0.05). Studies have also shown a significant reduction in systolic and diastolic blood pressures after Zinc supplementation. This first comprehensive systematic review and meta-analysis on the effects of Zinc supplementation in patients with diabetes demonstrates that Zinc supplementation has beneficial effects on glycaemic control and promotes healthy lipid parameters. Further studies are required to identify the exact biological mechanisms responsible for these results.

Effect of improved zinc status on T helper cell activation and TH1/TH2 ratio in healthy elderly individuals
            (Kahmann et al., 2006) Download
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.

The pigmentation of human iris influences the uptake and storing of zinc
            (Kokkinou et al., 2004) Download
Age-related macular degeneration (AMD) is more prevalent among the elderly Caucasians than in Africans. A significant association between light iris colour, fundus pigmentation and incidence of AMD is reported, suggesting a possible correlation with melanin pigment. Zinc is known to bind to melanin in pigmented tissues and to enhance antioxidant capacity by function as a cofactor or gene expression factor of antioxidant enzymes in the eye. In this in vitro study, we investigated the uptake and storage of zinc in human irides. Irides of blue and brown human eyes were used. The number of melanocytes was measured. Tissues without any treatment served as controls. The irides were incubated with 100 microM zinc chloride in culture medium for 24 h. Specimens of the tissues were stored for the uptake examination. The remained pieces were further incubated for 3 and 7 d to investigate the storage of zinc. The concentration of zinc was measured by inductively coupled plasma mass spectrometry (ICP-MS). Melanocytes count was significantly higher in the brown tissues (P < 0.0001). Zinc concentration of blue coloured irides after 24 h zinc treatment was close to the controls. We did not observe any significant storing. In contrast, the concentration of zinc in brown irides was significantly increased after 24 h (P < or = 0.01) and remained at a high level for 7 d. The uptake of zinc is likely dependent on the amount of pigmentation in human iris. Therefore, we assume that in patients suffering from AMD the degree of pigmentation of the irides and eventually fundi should be under consideration when the patients are treated with zinc supplementation.

Effects of zinc supplementation on cognitive function in healthy middle-aged and older adults: the ZENITH study
            (Maylor et al., 2006) Download
A randomised double-blind placebo-controlled design was employed to investigate the effects of Zn supplementation on cognitive function in 387 healthy adults aged 55-87 years. Several measures of visual memory, working memory, attention and reaction time were obtained using the Cambridge Automated Neuropsychological Test Battery at baseline and then after 3 and 6 months of 0 (placebo), 15 or 30 mg Zn/d. Younger adults (< 70 years) performed significantly better on all tests than older adults (> 70 years), and performance improved with practice on some measures. For two out of eight dependent variables, there were significant interactions indicating a beneficial effect (at 3 months only) of both 15 and 30 mg/d on one measure of spatial working memory and a detrimental effect of 15 mg/d on one measure of attention. Further work is required to establish whether these findings generalise to older adults in poorer mental and physical health and with less adequate Zn intake and status than the present sample.

Zinc in cancer prevention
            (Prasad et al., 2009) Download
Essentiality of zinc for humans was discovered 45 yr ago. Deficiency of zinc is prevalent world wide in developing countries and may affect nearly 2 billion subjects. The major manifestations of zinc deficiency include growth retardation, hypogonadism in males, cell-mediated immune dysfunctions, and cognitive impairment. Zinc not only improves cell mediated immune functions but also functions as an antioxidant and anti-inflammatory agent. Oxidative stress and chronic inflammation have been implicated in development of many cancers. In patients with head and neck cancer, we have shown that nearly 65% of these patients were zinc deficient based on their cellular zinc concentrations. Natural killer (NK) cell activity and IL-2 generation were also affected adversely. Th2 cytokines were not affected. In our patients, zinc status was a better indicator of tumor burden and stage of disease in comparison to the overall nutritional status. Zinc status also correlated with number of hospital admissions and incidences of infections. NF-kappa B is constitutively activated in many cancer cells, and this results in activation of antiapoptotic genes, VEGF, cyclin DI, EGFR, MMP-9 and inflammatory cytokines. Zinc inhibits NF-kappa B via induction of A-20. Thus, zinc supplementation should have beneficial effects on cancer by decreasing angiogenesis and induction of inflammatory cytokines while increasing apoptosis in cancer cells. Based on the above, we recommend further studies and propose that zinc should be utilized in the management and chemoprevention of cancer.

Dietary zinc supplementation during pregnancy prevents spatial and object recognition memory impairments caused by early prenatal ethanol exposure
            (Summers et al., 2008) Download
Alcohol-induced zinc (Zn) deficiency is one of the mechanisms proposed as a cause of ethanol teratogenicity. Subcutaneous Zn treatment with ethanol in early pregnancy has been shown to prevent birth abnormalities and memory impairments in mice. This study examined whether dietary Zn supplementation throughout pregnancy can prevent cognitive impairments caused by early ethanol exposure. Pregnant C57BL/6J mice were fed either a control (35 microg Zn/g) or Zn-supplemented (200 microg Zn/g) diet throughout pregnancy. On gestational day (GD) 8, mice received two intraperitoneal injections (4h apart) of either saline or 25% ethanol (0.015 mL/g). All offspring were screened for physical and behavioural defects (e.g. growth, visual, exploratory, anxiety, motor deficits). Twenty-four phenotypically-normal offspring were randomly selected from each of the four treatment groups (saline +/- Zn-supplementation, ethanol +/- Zn-supplementation) and tested at 60 d of age using a cross-maze escape task for spatial learning and memory impairments, and an object recognition task. While no differences were observed between treatments for spatial learning, offspring exposed to ethanol demonstrated spatial memory impairments at both 12 and 28 d after learning an escape task, with less correct trials and increased escape latency scores compared with saline-treated mice. Furthermore, these mice also exhibited impairments in object recognition memory. In comparison, ethanol-exposed offspring from dams fed a Zn-supplemented diet throughout pregnancy did not display spatial memory or object recognition deficits, performing at the same level as saline-treated offspring. Therefore, dietary Zn-supplementation during pregnancy prevents spatial and object recognition memory impairments caused by ethanol exposure during early pregnancy.

Zinc supplementation improved cognitive performance and taste acuity in Indian adolescent girls
            (Tupe and Chiplonkar, 2009) Download
OBJECTIVE: To test the efficacy of zinc supplementation through diet or ayurvedic zinc tablet on cognitive function and taste acuity in adolescent girls. METHODS: Using zinc-rich food items, snacks were prepared by adopting food-processing methods that enhance zinc bioavailability. Ayurvedic zinc tablet (jasad bhasma) was chosen as a natural elemental zinc supplement. Efficacy of snacks and the tablet was assessed in 180 schoolgirls (12.5 +/- 0.85 years) from Pune City, India, who were randomly allocated to any of the 3 groups: (1) ayurvedic zinc tablet-J, (2) zinc-rich snacks-D, or (3) Control-C. Supplementation was given on every school day (6 days/wk) for 10 weeks. All measurements were recorded at baseline and at the end of the study period. Food intake was recorded by 24-hour diet recall on 3 random days. Hemoglobin, serum ferritin, and plasma zinc were estimated on a fasting blood sample. Cognitive assessment was done on each participant using tests for simple reaction time (SRT), recognition reaction time (RRT), visual memory, and Raven's Progressive Matrices (RPM). Taste acuity was determined by recognition thresholds for salt (RTS) over a range of 10 different salt concentrations. RESULTS: A higher increase in plasma zinc (61.3%) was observed in the J group than in the D group (9.9%) (p < 0.01), whereas plasma zinc declined in the control group (-2.2%) over baseline (p > 0.1). Hemoglobin showed no change in all 3 groups (p > 0.1). Percent increment in scores for memory and RPM was significantly more in the D and J groups (24.5%-29.6%) than in the C group (6.5%) (p < 0.05). Mean SRT and RRT were reduced more in the D and J groups (5%-16%) than in the C group (1.6%) (p < 0.05). A significant fall in median RTS from 5 to 2.5 mmol/L was noted after both diet and zinc supplementation (p < 0.01); however, it remained the same at 5 mmol/L in the Control group after 10 weeks. CONCLUSION: Supplementation of ayurvedic zinc and zinc-rich foods are effective in improving cognitive performance and the recognition threshold for salt of adolescent girls.

 


References

Brewer, G. J. (2012), ‘Copper excess, zinc deficiency, and cognition loss in Alzheimer’s disease’, Biofactors, 38 (2), 107-13. PubMedID: 22438177
Capdor, J., et al. (2013), ‘Zinc and glycemic control: a meta-analysis of randomised placebo controlled supplementation trials in humans’, J Trace Elem Med Biol, 27 (2), 137-42. PubMedID: 23137858
Chasapis, C. T., et al. (2012), ‘Zinc and human health: an update’, Arch Toxicol, 86 (4), 521-34. PubMedID: 22071549
Dani, V., et al. (2007), ‘Chemopreventive potential of zinc in experimentally induced colon carcinogenesis’, Toxicol Lett, 171 (1-2), 10-18. PubMedID: 17590543
Evans, J. R. and J. G. Lawrenson (2012), ‘Antioxidant vitamin and mineral supplements for slowing the progression of age-related macular degeneration’, Cochrane Database Syst Rev, 11 CD000254. PubMedID: 23152201
Fischer Walker, C. and R. E. Black (2004), ‘Zinc and the risk for infectious disease’, Annu Rev Nutr, 24 255-75. PubMedID: 15189121
Haase, H., E. Mocchegiani, and L. Rink (2006), ‘Correlation between zinc status and immune function in the elderly’, Biogerontology, 7 (5-6), 421-28. PubMedID: 16953331
Jayawardena, R., et al. (2012), ‘Effects of zinc supplementation on diabetes mellitus: a systematic review and meta-analysis’, Diabetol Metab Syndr, 4 (1), 13. PubMedID: 22515411
Kahmann, L., et al. (2006), ‘Effect of improved zinc status on T helper cell activation and TH1/TH2 ratio in healthy elderly individuals’, Biogerontology, 7 (5-6), 429-35. PubMedID: 16967204
Kokkinou, D., et al. (2004), ‘The pigmentation of human iris influences the uptake and storing of zinc’, Pigment Cell Res, 17 (5), 515-18. PubMedID: 15357838
Maylor, E. A., et al. (2006), ‘Effects of zinc supplementation on cognitive function in healthy middle-aged and older adults: the ZENITH study’, Br J Nutr, 96 (4), 752-60. PubMedID: 17010236
Prasad, A. S., et al. (2009), ‘Zinc in cancer prevention’, Nutr Cancer, 61 (6), 879-87. PubMedID: 20155630
Summers, B. L., et al. (2008), ‘Dietary zinc supplementation during pregnancy prevents spatial and object recognition memory impairments caused by early prenatal ethanol exposure’, Behav Brain Res, 186 (2), 230-38. PubMedID: 17884190
Tupe, R. P. and S. A. Chiplonkar (2009), ‘Zinc supplementation improved cognitive performance and taste acuity in Indian adolescent girls’, J Am Coll Nutr, 28 (4), 388-96. PubMedID: 20368377