Type 2 diabetes mellitus (DM) is a major public health concern worldwide. In Hong Kong, all age prevalence of type 2 DM is around 11%, and the prevalence approaches 25% in those older than 65 years.1 Serious micro- and macrovascular complications are common in type 2 DM, and diabetic retinopathy (DR) is a major cause of vision loss.2,3 At a clinical population level, the risk of retinopathy is increased by poor glycemic control, increased blood pressure (BP), and elevated lipids.3–5 However, at the individual level, it is not currently possible to predict who will develop retinopathy, even though these “traditional” risk factors are monitored on a regular basis in type 2 DM patients. The lack of predictive biomarkers prevents early, cost-effective measures to target those who might benefit most from intensive therapy. There is also a lack of biomarkers that are sensitive indicators of early stages of complication onset.
Increased oxidative stress and depleted antioxidant status are often found in DM patients, and these may play an important role in the development of diabetic complications including retinopathy.6,7 The changes in oxidant:antioxidant balance associate with hyperglycemia, but the association is not straightforward, and it is not yet known whether increased oxidative stress predates, accompanies, or is a late consequence of metabolic complications of diabetes.6 We suggest that a wider biomarker profile, incorporating measures of oxidative stress and antioxidant balance as well as more traditional markers, may prove useful in identifying individuals at higher risk of developing complications or who are at the very early, potentially reversible, stage of complication development.
DR has a prevalence of almost 80% in those who have had type 2 DM for ≥20 years.8 The Early Treatment Diabetic Retinopathy Study (ETDRS) grading system categorizes diabetic fundus changes into five groups: no DR, questionable DR, early DR, non-proliferative DR (NPDR), and proliferative DR (PDR).9 Progression from early to advanced stages of DR has been thoroughly documented using this ETDRS grading system.9 Progression of DR can be arrested if treated during its early stages, but is often fairly advanced in many patients by the time of diagnosis of type 2 DM. Intensive therapy has been shown to decrease risk of DR progressing to clinically significant levels by 76%.10–12 However, intensive therapy is expensive, carries with it a significant risk of hypoglycemia, and for some patients the risk outweighs the benefit.3,13 From a public health perspective, it is more effective and economically feasible if high-risk individuals could be targeted for early and intensive treatment.12–15 However, and as noted above, these individuals cannot be identified based on currently available knowledge. This study aims to investigate associations between antioxidant status, oxidative stress, and traditional risk factors in relation to DR in type 2 DM subjects. The ultimate goal of this work is to establish a biomarker profile that will identify high-risk individuals or those in the early stages of DR. It is this group that would benefit most from early, intensive treatment, with preservation of visual function. Here, we present results of a cross-sectional descriptive study investigating interrelationships between biomarkers of glycemic control, lipids, and antioxidant/oxidative balance and their association with concurrent DR status.
Type 2 DM subjects were recruited from a local hospital-based diabetes clinic during August 2002 to February 2005. All participants were of Chinese ethnicity and had been diagnosed at least 1 year prior with type 2 DM, as defined by World Health Organization.16 Tenets of the Declaration of Helsinki were followed and institutional review board approval was granted for the study by both The Hong Kong Polytechnic University Research Ethics Sub-Committee and the Our Lady of Maryknoll Hospital Institutional Review Board. Patients older than 75 years, current smokers, or regular users of vitamin supplements were excluded. Volunteers were excluded if their medical records showed history of cardiovascular or renal complications or stroke, or if they had been previously referred to an ophthalmologist by the clinician at the diabetic clinic. A total of 632 patients volunteered, of whom 420 met these entrance criteria. All 420 reported being in good general health, apart from their diabetes.
Height and weight, waist:hip ratio (WHR), and BP were measured at entry. DR and other anterior and posterior ocular segment pathology were evaluated using (1) slitlamp biomicroscopy, (2) the Nidek anterior segment Scheimpflug camera (EAS-1000), and (3) 45° Topcon TRC NW6S fundus camera by an optometrist. Pupils were dilated with Mydrin-P and nine fundus photographs were taken in each eye after maximum dilation in nine fields of gaze: temporal; superior temporal; superior; superior nasal; nasal; inferior nasal; inferior; inferior temporal; and central posterior pole. Imagenet 2000 software (Tokyo, Japan) was used for image archiving. These photographs were arranged in an order to cover a full field of the retina and all were graded, masked to a standard photograph provided by the Fundus Reading Center (1991). Every retinopathy lesion was assessed by an optometrist following the grading rule described by the ETDRS Research Group.9 DR severity grading of background DR, NPDR, and PDR were defined as ETDRS scores of 14 to 20, 35 to 53, and 61 to 85, respectively. An ETDRS score of <14 translates to “no retinopathy detected.”
Fasting venous blood was collected for determination of glycemic control [fasting plasma glucose (FPG)] and glycosylated hemoglobin (HbA1c), plasma lipids [triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol], plasma uric acid, and biomarkers of oxidative stress and antioxidant status (lymphocytic DNA) damage using the comet assay, plasma allantoin, total antioxidant capacity (as the FRAP assay), ascorbic acid (vitamin C), α-tocopherol (“vitamin E,” which was standardized to plasma lipid content) and bilirubin, erythrocyte superoxide dismutase and glutathione peroxidase, and all biomarkers of antioxidant:oxidant balance. A spot urine sample was also collected for determination of urine microalbumin (a marker of early renal complications) and F2 isoprostanes (an additional marker of oxidative stress). Because the water content of urine can vary greatly, urine creatinine concentrations were determined to concentration-standardized urine microalbumin and F2 isoprostanes results.
Descriptive statistics were performed on the demographic and biomarker data of the 420 subjects recruited. These are presented as mean, standard deviation (SD), median, and range. Interrelationships between the various biomarkers and demographic data were investigated using Spearman bivariate correlation analysis. Because of the multiple correlations performed, only those correlation values reaching a level of p < 0.0001 were regarded as significant. On the basis of the eye tests, each patient was allocated to one of four groups: group 0 (No DR), group 1 [background DR (minimal retinal changes)], group 2 (NPDR), and group 3 (PDR), depending on the DR severity grading of the more badly affected eye. Kruskal–Wallis one-way analysis of variance was used for comparison of biomarker and demographic data across the DR severity grading groups with Dunn posttest. GraphPad Prism version 5.00 for Windows, (GraphPad Software, San Diego, CA) was used to perform all statistical tests.
Of the 420 subjects, 46 (11.0%) patients had no DR, 161 (38.3%) had background DR, 207 (49.3%) had NPDR, and 6 (1.4%) had PDR. This gives an overall DR prevalence of 89% in this group of apparently well type 2 DM patients in Hong Kong. Demographic and biomarker data on these patients at entry are presented in Table 1, along with typical values expected in healthy, non-diabetic adults. It can be seen that of the traditional HbA1c, Blood pressure, LDL-C (ABC) risk factors for complication development, glycemic control covered a wide span (HbA1c across the group ranged between 5.0 and 14.3%, and FPG between 3.4 and 15.3 mM). Antioxidant and oxidative stress biomarkers also varied widely. Average values of BP and plasma lipids (cholesterol and triglycerides) were similar to those found in healthy adults. However, average values mask the findings of very high BP, triglycerides and total cholesterol, and very low levels of the protective HDL cholesterol in some subjects. To investigate whether the subjects with increasingly severe DR were those with the poorest biomarker profile, the variables were compared across the four groups of DR severity grading. However, no significant differences in any of the demographic measures or biomarkers were found between any of the groups (Table 2).
In regard to interrelationship between the various biomarkers and demographics in these 420 patients, there were no strong associations found. There were, as expected, significant (p < 0.0001) direct correlations between age and systolic BP (ρ = 0.29), age and WHR (ρ = 0.21), WHR and plasma triglycerides (ρ = 0.19), WHR and uric acid (ρ = 0.30), and between the two dietary derived antioxidant vitamins, C and E, (ρ = 0.22) exist. There were also significant correlations (p < 0.0001) between the biomarkers of glycemic control and a biomarker of oxidative stress (DNA damage): HbA1c and DNA damage (ρ = 0.32); FPG and DNA damage (ρ = 0.52). DNA damage was also significantly and inversely correlated (p < 0.0001) with both plasma ascorbic acid (ρ = −0.41) and plasma antioxidant level corrected for uric acid (ρ = −0.21). It was also found that those with higher triglycerides had lower HDL cholesterol (ρ = −0.34; p < 0.0001), and that those with higher total cholesterol had lower lipid standardized vitamin E (ρ = −0.22; p < 0.0001). There was an unexpected significant (ρ = 0.33) p < 0.0001) direct correlation between lipid standardized vitamin E (an antioxidant) and allantoin (a biomarker of oxidative stress).
DR is a major and potentially preventable or reversible (in its early stages) complication of type 2 DM. However, beyond a certain stage DR progression is relentless, and this “retinopathic momentum” carries on regardless of intervention.10,17 Figures for DR prevalence vary across countries and groups (Table 3). In Hong Kong, prevalence in 6165 type 2 DM patients from 10 primary care clinics was reported to be 28% (background DR, 13.0%, NPDR, 14.8%, PDR, 0.5%),18 and a recently published study of 413 type 2 DM patients found that 162 (39%) had some degree of DR [134 (32%) had NPDR and 28 (7%) had PDR, with 26 (6%) having macular edema].4 The prevalence of any kind of DR in this study of 420 apparently well type 2 DM patients was almost 90%: 38% had background DR, 49% had NPDR, and <2% had PDR. These figures are disturbingly high but may be a more true reflection of prevalence. Among previous studies in Hong Kong, DR grading was performed by non-mydriatic means using only one or two centered 45° fundus photographs rather than the full nine field of gaze adopted in this study. This more sensitive and comprehensive method of testing may reveal otherwise undetectable background and early NPDR.
The very high prevalence of previously undetected DR found in this study was both unexpected and alarming. Poor glycemic control has been reported to be predictive of development of DR in type 2 DM4,9 and risk of its occurrence and progression to PDR can be significantly lowered by tight glycemic control, especially with early and intensive treatment.10–13 In addition, intensive lipid treatment, with a combination of fenofibrate and simvastatin, has been shown recently to retard the progression of DR. This may constitute a therapeutic advantage particularly in those who cannot achieve optimal glycemic control.5 Interestingly, in this study, the six newly discovered subjects with PDR did not have poorer glycemic control, or show higher lipids or BP, nor did they have lower antioxidant balance or have higher oxidative stress levels than those with no evidence of DR. Indeed, there were no significant differences seen in any of the demographics or biomarkers measured across the four DR categories (PDR, NPDR, early, or no retinal involvement), even though a wide range of biomarkers, incorporating both traditional and candidate biomarkers of risk was investigated. In particular, no differences were seen in HbA1c levels, which averaged 7.6% in both DR and non-DR groups. This is in contrast to a previous study,4 which showed that baseline HbA1c levels were higher (averaging 8.5%) in those who had DR compared with those who did not (HbA1c average 7.8%). In a follow-up arm of that study, 43 (20%) of the 212 type 2 DM patients who were free of DR at baseline developed DR (ETDRS grading of 21 or greater in either eye) in the next 3 to 5.6 years (mean follow-up of 4.2 years). A high baseline HbA1c was found to be the only predictor on multivariate analysis, being significantly higher in the progressors compared with those who remained free of DR [mean (SD) 8.6 (1.7)% vs. 7.7 (1.3)%; p < 0.0005]. Of the 121 patients found to have NPDR at baseline and who were available for follow-up, 42 (35%) showed a two-step or greater progression in the ETDRS grading during follow-up.4 Baseline HbA1c and FPG levels were significantly higher in those who progressed compared with those who did not [mean (SD) 9.1(1.3)% vs. 8.2(1.4%) for HbA1c, and 10.2(3.1) mM and 9.0(2.4) mM for FPG]. Systolic BP and the prevalence of macroalbuminuria at baseline were also significantly higher in the DR progressors (p < 0.001).
These findings of Tam et al.4 reinforce the importance of good glycemic control in preventing or delaying progress of DR. However, the data also show that there is considerable overlap in HbA1c levels in those who go on to develop DR compared with those who do not. There is also wide overlap in traditional biomarkers between those whose DR remains stable in its early stages and those in whom the retinopathic momentum has already set in. Therefore, although HbA1c is probably the strongest single biomarker for DR risk, elevated HbA1c is clearly not predictive of DR development or progression at the individual level. For this reason, further investigation into the factors that are involved in development and progression of DR is needed. One candidate is oxidative stress, and in this regard, the measurement of loss of alpha crystallins in the lens as a marker for oxidative stress damage in the eye19 could be a useful approach, albeit indirectly reflecting the retina. Another additional biomarker of relevance to the eye is measurement of aldose reductase, a key player in the polyol pathway. The activity of aldose reductase can be measured in red blood cells and may indirectly represent activity and polyol levels in retinal tissues.20
In conclusion, up to 60% of type 2 DM patients will have developed some degree of retinopathy by 10 years since their diagnosis, and this rises to >80% after 15 years of DM.21 Of those with established DR, some will remain in the early stages, but the DR momentum of others will build and take them into the irreversible, vision threatening stages. HbA1c is known to be an important factor in DR development and progression, but it is not an accurate predictor of either in individual patients. In this study of 420 type 2 DM subjects, DR prevalence was found to be very high, but no biomarker differences, including in HbA1c, were seen in those with DR compared with those free of DR. In the phase II follow-up part of this study, we will investigate if the extended biomarker profile, incorporating biomarkers of antioxidant status and oxidative stress as well as more traditional markers of ABC control can help identify those individuals who are at higher risk of incident DR, or those who will show more rapid progression from NPDR to PDR.
We thank the research team and nurses at Our Lady of Maryknoll Hospital Diabetes Education and Management Centre for their kind assistance with the study.
This research was funded by The Hong Kong Polytechnic University Grants A-PD86 to CSY Lam and BD02 to IFF Benzie.
Carly S.Y. Lam
School of Optometry
The Hong Kong Polytechnic University
Hung Hom, Hong Kong
SAR of China
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Keywords:© 2011 American Academy of Optometry
retinopathy; type 2 diabetes; oxidative stress; antioxidants