EAL

FNOA: Antioxidants (2011-2012)

Citation:

Study Design:

Class:

- Click here for explanation of classification scheme.

Quality Rating:

Research Purpose:

To measure zinc and copper levels in the retinal pigment epithelium (RPE) and choroid complex and in the neural retina in subjects with and without age-related macular degeneration (AMD).

Inclusion Criteria:

Eye tissue from human donors at the time of autopsy with a reported history of AMD.

Exclusion Criteria:

Eye tissue from human donors at the time of autopsy with previous ocular trauma, intraocular tumor glaucoma, diabetic retinopathy, or any systemic disease or occupation likely to cause aberration in metal content.

Description of Study Protocol:

Recruitment

Eye tissue from human donors at the time of autopsy with a reported history of AMD.

Design

Case-Control Study

Blinding used

Not used

Intervention

Oral multivitamin supplementation was used in 13 of the 44 of the subjects, not used on 23 subjects and not recorded in eight subjects.

Statistical Analysis

Metal levels from two eyes of the same subject were averaged and treated as one observation
Tissue metal levels, as a dry weight were compared using Wilcoxon rank-sum test
Correlations between age and metal levels were examined by using Pearson product moment analysis.

Data Collection Summary:

Timing of Measurements

The mean time from death to tissue dissection ± standard deviation was 131.1±3.9 hours.

Dependent Variables

Copper and zinc in the retina
Copper and zinc in the RPE/choroid complex.

Independent Variables

RPE and choroid complex classified into Stages 1 to 4 based on Minnesota Grading System (MGS) as defined by the Age Related Eye Disease Study: Stage 1 (subjects without AMD), Stage 2 (early AMD with pigment changes and small drusen), Stage 3 (intermediate AMD with intermediate size drusen, a single large drusen or noncentral geographic atrophy) or Stage 4 (advanced AMD with central geographic atrophy or choroidal neovascularization)
History of multivitamin supplementation
Age.

Control Variables

None.

Description of Actual Data Sample:

Initial N: 88 donor eyes from 44 subjects
- 13 men and 16 women in the control group
- Seven men and eight women in the AMD group
Attrition (final N): No attrition reported
Age:
- 29 subjects with a mean age ± SD 73±12 years were analyzed in the control group
- 15 subjects with a mean age ± SD 78±10 years were analyzed in the AMD group (MGS stages 2 through 4)
Ethnicity: Not specified
Other relevant demographics: Of the 30 eyes (15 subjects) in the AMD group
- Four eyes (13%) were MGS stage 2
- 19 eyes (64%) were MGS stage 3
- Four eyes (23%) were MGS stage 4
Anthropometrics: Cases and controls were not matched for age
Location: Mayo Clinic, Rochester, MN.

Summary of Results:

Other Findings

Mean zinc and copper levels in the RPE and choroid complex of subjects with AMD were reduced 24% and 23% respectively when compared with those of subjects without AMD. (P=0.01 and P=0.002).
Although zinc and copper levels in the neural retina in subjects with AMD were lower than those in subjects without AMD, a significant difference was not detected. (P=0.09 and P=0.69).
Zinc and copper levels were correlated in the neural retina and RPE and choroid complex. No correlation between age and RPE and choroid complex zinc levels (r=0.32, P=0.26) and between age and RPE and choroid complex copper levels (r=0.19, P=0.51) was found.
Zinc levels in the RPE and choroid complex (288.3±84.6µg/g) and retina (114.5±22.6µg/g) in subjects using multivitamin supplementation were not different than levels in the RPE and choroid complex (270.1±105.3µg/g) and retina (125.6±69.2µg/g) of subjects not taking multivitamins (P=0.37 and P=1).
The minimum detectable difference (β=0.80 and α=0.05) for zinc in the RPE and choroid complex and retina was 99 and 57µg/g
Copper levels in the RPE and choroid complex (6.3±1.4µg/g) and retina (8.8±1.3µg/g in subjects using multivitamin supplementation were not different than levels in the RPE and choroid complex (6.2±1.4µg/g) and retina (9.5±3.8µg/g) of subjects not taking multivitamins (P=0.82 and P=0.55)
The minimum detectable difference for copper levels in the RPE and choroid complex and retina was 1.5 and 2.8µg/g.

Author Conclusion:

The results of this study showing decreased zinc and copper in the RPE and choroid complex of AMD-affected subjects supports the hypothesis that metal dysregulation may be involved in the pathogenesis of AMD. Although a relationship exists between AMD and reduced RPE and choroid zinc and copper levels, it is not necessarily a casual relationship. Further study is needed of the mechanisms of zinc and copper homeostasis in healthy human retina and in AMD pathogenesis.

Funding Source:

University/Hospital:

Mayo Clinic

Not-for-profit

Mayo Foundation

Foundation associated with industry:

Other:

Research To Prevent Blindness, Inc.

Reviewer Comments:

The authors noted the following limitations:

Power limitations contributed to an inability to detect differences when analyzing multivitamin supplementation
The analysis was limited by the lack of age-matched controls. Age is a primary risk factor for AMD, however, no significant difference was found in age between study subjects with and without AMD.
Limited tissue availability resulted in small sample sizes which reduced power estimates and precluded multivariate analysis.
Although, reduced zinc and copper levels in the RPE and choroid complex were associated with AMD, it is not possible to know whether the relationship is truly one of cause and effect.

Quality Criteria Checklist: Primary Research
Relevance Questions
	1.	Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies)	Yes
	2.	Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about?	Yes
	3.	Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice?	Yes
	4.	Is the intervention or procedure feasible? (NA for some epidemiological studies)	Yes

Validity Questions
1.	Was the research question clearly stated?		Yes
	1.1.	Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified?	Yes
	1.2.	Was (were) the outcome(s) [dependent variable(s)] clearly indicated?	Yes
	1.3.	Were the target population and setting specified?	Yes
2.	Was the selection of study subjects/patients free from bias?		Yes
	2.1.	Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study?	Yes
	2.2.	Were criteria applied equally to all study groups?	Yes
	2.3.	Were health, demographics, and other characteristics of subjects described?	Yes
	2.4.	Were the subjects/patients a representative sample of the relevant population?	Yes
3.	Were study groups comparable?		???
	3.1.	Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT)	Yes
	3.2.	Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline?	No
	3.3.	Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)	Yes
	3.4.	If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis?	N/A
	3.5.	If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.)	???
	3.6.	If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")?	N/A
4.	Was method of handling withdrawals described?		N/A
	4.1.	Were follow-up methods described and the same for all groups?	N/A
	4.2.	Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.)	N/A
	4.3.	Were all enrolled subjects/patients (in the original sample) accounted for?	N/A
	4.4.	Were reasons for withdrawals similar across groups?	N/A
	4.5.	If diagnostic test, was decision to perform reference test not dependent on results of test under study?	N/A
5.	Was blinding used to prevent introduction of bias?		Yes
	5.1.	In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate?	N/A
	5.2.	Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.)	Yes
	5.3.	In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded?	N/A
	5.4.	In case control study, was case definition explicit and case ascertainment not influenced by exposure status?	Yes
	5.5.	In diagnostic study, were test results blinded to patient history and other test results?	N/A
6.	Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described?		No
	6.1.	In RCT or other intervention trial, were protocols described for all regimens studied?	N/A
	6.2.	In observational study, were interventions, study settings, and clinicians/provider described?	N/A
	6.3.	Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect?	???
	6.4.	Was the amount of exposure and, if relevant, subject/patient compliance measured?	???
	6.5.	Were co-interventions (e.g., ancillary treatments, other therapies) described?	???
	6.6.	Were extra or unplanned treatments described?	???
	6.7.	Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups?	N/A
	6.8.	In diagnostic study, were details of test administration and replication sufficient?	N/A
7.	Were outcomes clearly defined and the measurements valid and reliable?		Yes
	7.1.	Were primary and secondary endpoints described and relevant to the question?	Yes
	7.2.	Were nutrition measures appropriate to question and outcomes of concern?	Yes
	7.3.	Was the period of follow-up long enough for important outcome(s) to occur?	Yes
	7.4.	Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures?	Yes
	7.5.	Was the measurement of effect at an appropriate level of precision?	Yes
	7.6.	Were other factors accounted for (measured) that could affect outcomes?	Yes
	7.7.	Were the measurements conducted consistently across groups?	Yes
8.	Was the statistical analysis appropriate for the study design and type of outcome indicators?		???
	8.1.	Were statistical analyses adequately described and the results reported appropriately?	Yes
	8.2.	Were correct statistical tests used and assumptions of test not violated?	Yes
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	Yes
	8.4.	Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)?	N/A
	8.5.	Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)?	???
	8.6.	Was clinical significance as well as statistical significance reported?	Yes
	8.7.	If negative findings, was a power calculation reported to address type 2 error?	No
9.	Are conclusions supported by results with biases and limitations taken into consideration?		Yes
	9.1.	Is there a discussion of findings?	Yes
	9.2.	Are biases and study limitations identified and discussed?	Yes
10.	Is bias due to study's funding or sponsorship unlikely?		Yes
	10.1.	Were sources of funding and investigators' affiliations described?	Yes
	10.2.	Was the study free from apparent conflict of interest?	Yes