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Clinical and Experimental Ophthalmology 2014; 42: 841–845 doi: 10.1111/ceo.12329
Original Article Increased D-serine in the aqueous and vitreous humour in patients with proliferative diabetic retinopathy Haiyan Jiang MS,* Jinlin Du MD,* Tao He MB, Jia Qu MD MS, Zongming Song MD PhD and Shengzhou Wu MD PhD School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, and State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
ABSTRACT
the blood and in the vitreous specimens from PDR were measured with spectrophotometry to correct possible introduction of amino acids from PDR haemorrhage.
Background: The objective of this clinical study is to examine the association between D-serine and diabetic retinopathy (DR). Design: Retrospective, case-control study was performed in the affiliated Eye Hospital of Wenzhou Medical University. Participants: This study included 25 patients with proliferative diabetic retinopathy (PDR), and 25 sexand age-matched control subjects, i.e. patients with idiopathic macular hole and idiopathic epiretinal membrane. Methods: Clinical diagnoses were made by the senior ophthalmologists in the Eye Hospital; the aqueous and vitreous humour specimens were collected from these patients undergoing pars plana vitrectomy for treating complications. Main Outcome Measures: The aqueous and vitreous levels of D-serine and glutamate were measured with reverse-phase high-performance liquid chromatography (HPLC); the contents of haemoglobin in
Results: The concentrations of D-serine in the aqueous or vitreous humour were significantly higher in patients with PDR compared with control subjects. The vitreous concentrations of D-serine in PDR were 25.55 ± 0.63 μmol/L compared with control subjects at 22.76 ± 0.36 μmol/L (P = 0.002); the levels of D-serine in the aqueous humour from patients with PDR were 29.08 ± 1.31 μmol/L compared with control subjects at 24.22 ± 0.65 μmol/L (P = 0.006). Correction from possible introduction of D-serine from the vitreous haemorrhage in PDR did not significantly alter the findings. Conclusions: Increased D-serine in the aqueous and vitreous humour was found in patients with PDR compared with control subjects. Key words: diabetic retinopathy, excitotoxicity, glutamate, retinal neuronal death, serine racemase.
■ Correspondence: Dr Shengzhou Wu and Dr Zongming Song, Key Laboratory of Visual Science, National Ministry of Health, School of Optometry and Ophthalmology, Wenzhou Medical University, Zhejiang Province, China. Email: [email protected] or [email protected] Received 25 January 2014; accepted 12 March 2014. Competing/conflicts of interest: No stated conflict of interest. Funding sources: The study was supported by National Natural Science Foundation of China (81371027), by Zhejiang Province Natural Science foundation (Y2110086), by Qianjiang Scholar Scheme (QJD1202020), by Chinese Ministry of Education (20133321120002), and by start-up funding (89210001) from Wenzhou Medical University to Dr Shengzhou Wu. *Jiang Haiyan and Du Jinlin contributed to the work equally. © 2014 Royal Australian and New Zealand College of Ophthalmologists
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INTRODUCTION
Table 1.
Diabetic retinopathy (DR) is a leading cause of adult blindness and one of the most common complications of diabetes. It becomes prevalent after about a decade of diabetes, ultimately leading to retinal oedema, neovascularization and vision loss in 5% of patients.1,2 The natural history of DR has been divided into an early, non-proliferative stage, and a later, proliferative stage. The pathology of DR involves inflammation-related changes in retinal vessels and neurodegeneration in the retinal ganglion cell layer (RGCL) and inner nuclear layer (INL);3 some evidence indicate this neuronal cell death precedes vascular changes in DR.3,4 Excitotoxins including homocysteine and glutamate induce apoptosis in RGC, which suggests that excitotoxicity is associated with this RGC degeneration.5 RGC degeneration in DR linking with excitotoxicity is substantiated with a line of evidence: increased retinal/or vitreous glutamate in patients with DR and in DR animal models;6–8 altered protein levels of ionotropic glutamate receptor subunits in rat and human diabetic retina;9,10 protection of retinal neuronal loss in diabetic rats3 by memantine, an uncompetitive antagonist of N-methyl-D-aspartate receptor (NMDAR). Recently, excitotoxicity contributing to neural degeneration was also linked to activity of serine racemase (SR), an enzyme that converts L-serine to D-serine.11–14 D-serine acts as a co-agonist at the glycineB site of the NMDAR thus influences neurotransmission and fine-tunes excitotoxicity.13–15 Evidence indicate that NMDA toxicity towards RGCs is enhanced by intravitreal injection of D-serine or glycine, whereas blocking glycine transport or blocking the glycineB binding site with 5, 7dichlorokynurenic acid (DCKA) reduces toxicity.16 Our prior results demonstrate that D-serine and glutamate are increased in the aqueous humour in the STZ-induced diabetic rats.17 In this study, the levels of D-serine and glutamate in the aqueous and/or vitreous humour in patients with PDR were determined to investigate their potential roles in retina neuronal damage.
Variable
Patients’ characteristics Patients with PDR (n = 25)
Patients with IMH or IME (n = 25)
58.6 ± 9.9
65.5 ± 7.1
13 (52%) 12 (48%)
10 (40%) 15 (60%)
1 (4%) 24 (96%)
0 (0) 0 (0)
Age, mean(SD), year Sex Male Female Categories of diabetes Type 1 Type II Duration of diabetes, year 5–10 11–20 21–30 Hypertension IOP, mean (SD), mmHg Retinal detachment Vitreous haemorrhage
3 (12%) 20 (80%) 2 (8%) 12 (48%) 13.4 ± 4.0 10 (40%) 17 (68%)
0 (0) 0 (0) 0 (0) 8 (32%) 13.6 ± 2.5 0 (0) 0 (0)
IME, idiopathic epiretinal membrane; IMH, idiopathic macular hole; IOP, intraocular pressure; PDR, proliferative diabetic retinopathy; SD, standard deviation. Unless otherwise indicated, data were given as the numbers of the participating patients and as the percentages in the corresponding groups, shown in parentheses.
idiopathic macular hole and idiopathic epiretinal membrane. The undiluted specimens were provided by the surgeons in the Retinal Surgery Department of Eye Hospital in Wenzhou Medical University. The characteristics of the patients were listed in Table 1. The patients with prior vitrectomy, intraocular ischaemia, and psychiatric problems18 were excluded from the study due to considering the causes other than diabetic retinopathy. PDR was defined with the presence of neovascularization, fibroproliferation of the disc or elsewhere on the retina under fundus examination and fluorescein angiography; macular hole with the presence of small break in macular, and epiretinal membrane with the presence of abnormal membrane that grows over macular area, which were examined with optical coherence tomography (OCT). All the diagnoses were made by the senior ophthalmologists in the Retinal Surgery Department of Eye Hospital in Wenzhou Medical University.
METHODS Patients
Sample collections
The study followed the tenets of the declaration of Helsinki and was approved by the ethics committee of Wenzhou Medical University. The informed consents were obtained from the subjects after explanation of the nature and the purposes of the study. The vitreous and aqueous humour specimens were collected from patients undergoing pars plana vitrectomy, including patients with PDR and patients with
Three-port, 23-gauge pars plana vitrectomy was conducted in which vitreous cutter was introduced via transconjunctival approach. A sample of undiluted vitreous (0.4–0.6 mL) was aspirated manually through suction port into a disposable tuberculin syringe set through a cannula and a sample of undiluted aqueous (0.1–15 mL) was also collected. Fasting venous blood samples were collected for
© 2014 Royal Australian and New Zealand College of Ophthalmologists
Role of D-serine in retinal ganglion cell death glucose, haemoglobin, glycated haemoglobin, and cholesterol assays.
Materials D-serine, glutamate, glacial acetic acid, o-phthaldialdehyde (OPA), and Boc-L-Cys were obtained from Sigma (St Louis, MO, USA). All solvents of HPLC grade were purchased from Merck (Darmstadt, Germany). Ultra-pure water prepared by a Milli-Q purification system from Millipore (Bedford, MA, USA) was used for preparing the mobile phase and all other solutions.
Determination of glutamate and D-serine Reverse-phase HPLC with fluorimetric detection was used to determine glutamate, D-serine as reported before.19 Pre-column derivatization with o-phthaldialdehyde (OPA) in combination with N-tert-butyloxycarbonyl-L-cysteine was used. A 3.5-μ column (150 × 4.6 mm, ZORBAX Eclipse AAA column) was used to separate the amino acids and the methods to establish the linear gradients in mobile phase followed the previous protocol.11 The categories of samples were blinded to the researchers (Jiang HY and Du JL) conducting the analyses.
Correction for vitreous haemorrhage The correction method followed the previous procedure.6 Haemoglobin concentration in blood was determined using automatic blood cell analyser (XT1800i; Sysmex Instruments, Sysmex, Kobe, Japan) (minimum level of detection, 1.0 g/dL) and in the vitreous using spectrophotometry (722S, Shanghai Precision & Scientific Instrument Co., Ltd, Shanghai, China) (minimum level of detection, 0.03 g/dL) to correct for the possible introduction of D-serine by vitreous haemorrhage. Corrected concentrations were calculated as follows:
[ D-serine] corrected = ([ D-serine] measured × [ Hgb] blood − [D-serine] blood × [ Hgb] vitreous) ([ Hgb] blood − [ Hgb] vitreous)
843 examined with the Pearson χ2 test. P < 0.05 was used as the criterion to reject the null hypothesis.
RESULTS Our results revealed the vitreous concentrations of D-serine (25.55 ± 0.63 μmol/L) in patients with PDR were significantly higher than patients with idiopathic macular hole and idiopathic epiretinal membrane with D-serine at 22.76 ± 0.36 μmol/L (P = 0.002) (Fig. 1); the aqueous concentrations of D-serine (29.08 ± 1.31 μmol/L) in patients with PDR were also significantly higher than control subjects at 24.22 ± 0.65 μmol/L (P = 0.006). We also revealed that the concentrations of glutamate in the aqueous and vitreous humour in patients with PDR were significantly higher than those in control subjects, which is consistent with previous report that the vitreous glutamate in patients with PDR is elevated.6 Our results indicated that the vitreous glutamate in patients with PDR was 12.23 ± 1.53 μmol/L compared with control subjects at 5.65 ± 0.48 μmol/L (P = 0.021) (Fig. 1); the aqueous glutamate in patients with PDR was 15.55 ± 1.67 μmol/L compared with control subjects at 11.22 ± 0.54 μmol/L (P = 0.021). To avoid the introduction of D-serine and glutamate from the vitreous haemorrhage from PDR, we adopted the correction strategy described in the Methods. The mean blood haemoglobin concentration in patients with PDR was 121.22 ± 17.18 g/L (mean ± SD) and the vitreous haemoglobin concentration in patients with PDR was 0.49 ± 0.18 g/L (mean ± SD) determined by spectrophotometry. Corrections did not alter the results for D-serine, for instance, the vitreous level of D-serine after correction was 25.55 ± 0.635 μmol/L compared to 25.55 ± 0.63 μmol/L before correction; the aqueous level of D-serine after correction was 29.09 ± 1.32 μmol/L compared with 29.08 ± 1.31 μmol/L before corrections. Similarly, the corrections for glutamate did not significantly alter the results, for instance, the aqueous and vitreous glutamate were 11.79 ± 1.54, 15.11 ± 1.67 μmol/L, respectively, after corrections, which were significantly higher than control subjects (P < 0.001 and P = 0.053).
DISCUSSION Statistic analyses All values were given as mean ± SEM, unless otherwise indicated. Since the values of D-serine and glutamate in the aqueous or vitreous did not have normal distributions, non-parametric Mann– Whitney U-test was used to compare the difference between PDR and control subjects. The distributions of age and sex in PDR and control subjects were
We hereby indicated increased D-serine in the aqueous and vitreous humour in patients with PDR compared with the patients with idiopathic macular hole and idiopathic epiretinal membrane. To our knowledge, this is the first report to probe D-serine in the aqueous and vitreous humour in patients with PDR. Since serine racemase expression declines with ageing,20 we carefully chose the patients without age
© 2014 Royal Australian and New Zealand College of Ophthalmologists
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Jiang et al. (b)
3 3
2
1
2
4
1 0 8
10
12
14 16 18 Elution Time (min)
(c) Fluorescent Intensity (LU)
Fluorescent Intensity (LU)
5
4
20
1 5
1 0 10
12
14 16 18 Elution Time (min)
20
3 2 1
1
10
12
30
14 16 18 Elution Time (min)
20
22
Contorl PDR
*
25 *
20 15 10 5
22
5
2
0
(d)
3
8
3
8
4
2
4
22
34
2
4
Content of Glu and D-se (μmol/L)
Fluorescent Intensity (LU)
(a)
D-serine
Glutamate
Figure 1. Increased D-serine and glutamate in the vitreous of patients with proliferative diabetic retinopathy (PDR), determined by high-performance liquid chromatography (HPLC). (a) Amino acid standards were separated by reverse-phase HPLC; 1: L-Asp, TR = 9.445 min; 2: L-Glu, TR = 13.45 min; 3: L-Ser, TR = 19.946 min; 4: L-Gln, TR = 20.578 min; 5: D-ser, TR = 21.752. Typical running scripts for the vitreous humour from control subjects were indicated in (b) or from patients with PDR in (c). Quantifications of D-ser and glutamate in control subjects and patients with PDR were indicated in (d). The results shown were mean ± SEM from 25 control subjects and 25 patients with PDR (*P < 0.05 vs. control).
difference between PDR and control subjects (P = 1). Simultaneously, the sex distribution in each group was also delicately designed, revealed by no difference existing between PDR and control subjects (P = 0.186). To exclude the interference from other ocular disorders, particularly with RGC death, i.e. glaucoma,21 in which RGC death may overlap the neuronal death occurring in DR, we excluded patients with high intraocular pressure (IOP) and the patients selected were without glaucoma (Table 1). Further, IOPs in patients with PDR were not different from those in control subjects (P = 0.079, Student t-test). We also collected aqueous humour from nonproliferative diabetic retinopathy patients (NPDR, n = 11) during the injections of anti-angiogenic medicines (Avastin or Lucentis) to the vitreous cavities. There were no differences on sex, age and IOP between NPDR and control subjects. The aqueous level of D-serine (32.19 ± 4.08 μmol/L), glutamate (15.66 ± 2.10 μmol/L) were significantly higher than those in control subjects (P = 0.006, 0.016, respectively). However, there were no difference in the levels of the aqueous D-serine and glutamate between patients with PDR and NPDR (P = 0.710, 0.761, respectively). In summary, our study demonstrated increased D-serine along with glutamate in the aqueous and
vitreous humour in patients with PDR, which suggests that enhanced NMDA-receptor activity is a contributing factor for RGC death occurring in DR.
ACKNOWLEDGEMENTS All the authors appreciate the dedication and patience from participants and the funding sources.
REFERENCES 1. Archer DB. Bowman Lecture 1998. Diabetic retinopathy: some cellular, molecular and therapeutic considerations. Eye (Lond) 1999; 13 (Pt 4): 497–523. 2. Cai J, Boulton M. The pathogenesis of diabetic retinopathy: old concepts and new questions. Eye (Lond) 2002; 16: 242–60. 3. Kusari J, Zhou S, Padillo E, Clarke KG, Gil DW. Effect of memantine on neuroretinal function and retinal vascular changes of streptozotocin-induced diabetic rats. Invest Ophthalmol Vis Sci 2007; 48: 5152–9. 4. Barber AJ. A new view of diabetic retinopathy: a neurodegenerative disease of the eye. Prog Neuropsychopharmacol Biol Psychiatry 2003; 27: 283–90. 5. Moore P, El-sherbeny A, Roon P, Schoenlein PV, Ganapathy V, Smith SB. Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine. Exp Eye Res 2001; 73: 45–57.
© 2014 Royal Australian and New Zealand College of Ophthalmologists
Role of D-serine in retinal ganglion cell death 6. Ambati J, Chalam KV, Chawla DK et al. Elevated gamma-aminobutyric acid, glutamate, and vascular endothelial growth factor levels in the vitreous of patients with proliferative diabetic retinopathy. Arch Ophthalmol 1997; 115: 1161–6. 7. Lieth E, Barber AJ, Xu B et al. Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy. Penn State Retina Research Group. Diabetes 1998; 47: 815–20. 8. Kowluru RA, Engerman RL, Case GL, Kern TS. Retinal glutamate in diabetes and effect of antioxidants. Neurochem Int 2001; 38: 385–90. 9. Santiago AR, Gaspar JM, Baptista FI et al. Diabetes changes the levels of ionotropic glutamate receptors in the rat retina. Mol Vis 2009; 15: 1620–30. 10. Santiago AR, Hughes JM, Kamphuis W, Schlingemann RO, Ambrosio AF. Diabetes changes ionotropic glutamate receptor subunit expression level in the human retina. Brain Res 2008; 1198: 153–9. 11. Wu SZ, Bodles AM, Porter MM, Griffin WS, Basile AS, Barger SW. Induction of serine racemase expression and D-serine release from microglia by amyloid betapeptide. J Neuroinflammation 2004; 1: 2. 12. Wu SZ, Jiang S, Sims TJ, Barger SW. Schwann cells exhibit excitotoxicity consistent with release of NMDA receptor agonists. J Neurosci Res 2005; 79: 638–43. 13. Inoue R, Hashimoto K, Harai T, Mori H. NMDA- and beta-amyloid1-42-induced neurotoxicity is attenuated in serine racemase knock-out mice. J Neurosci 2008; 28: 14486–91. 14. Mustafa AK, Ahmad AS, Zeynalov E et al. Serine racemase deletion protects against cerebral ischemia and excitotoxicity. J Neurosci 2010; 30: 1413–6.
845 15. Stevens ER, Esguerra M, Kim PM et al. D-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors. Proc Natl Acad Sci U S A 2003; 100: 6789–94. 16. Hama Y, Katsuki H, Tochikawa Y, Suminaka C, Kume T, Akaike A. Contribution of endogenous glycine site NMDA agonists to excitotoxic retinal damage in vivo. Neurosci Res 2006; 56: 279–85. 17. Jiang H, Fang J, Wu B et al. Overexpression of serine racemase in retina and overproduction of D-serine in eyes of streptozotocin-induced diabetic retinopathy. J Neuroinflammation 2011; 8: 119. 18. Hashimoto K, Fukushima T, Shimizu E et al. Decreased serum levels of D-serine in patients with schizophrenia: evidence in support of the N-methyl-D-aspartate receptor hypofunction hypothesis of schizophrenia. Arch Gen Psychiatry 2003; 60: 572–6. 19. Hashimoto A, Nishikawa T, Oka T, Takahashi K, Hayashi T. Determination of free amino acid enantiomers in rat brain and serum by highperformance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. J Chromatogr 1992; 582: 41–8. 20. Dun Y, Duplantier J, Roon P, Martin PM, Ganapathy V, Smith SB. Serine racemase expression and D-serine content are developmentally regulated in neuronal ganglion cells of the retina. J Neurochem 2008; 104: 970–8. 21. Kerrigan-Baumrind LA, Quigley HA, Pease ME, Kerrigan DF, Mitchell RS. Number of ganglion cells in glaucoma eyes compared with threshold visual field tests in the same persons. Invest Ophthalmol Vis Sci 2000; 41: 741–8.
© 2014 Royal Australian and New Zealand College of Ophthalmologists
Clinical and Experimental Ophthalmology 2014; 42: 841–845 doi: 10.1111/ceo.12329
Original Article Increased D-serine in the aqueous and vitreous humour in patients with proliferative diabetic retinopathy Haiyan Jiang MS,* Jinlin Du MD,* Tao He MB, Jia Qu MD MS, Zongming Song MD PhD and Shengzhou Wu MD PhD School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, and State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
ABSTRACT
the blood and in the vitreous specimens from PDR were measured with spectrophotometry to correct possible introduction of amino acids from PDR haemorrhage.
Background: The objective of this clinical study is to examine the association between D-serine and diabetic retinopathy (DR). Design: Retrospective, case-control study was performed in the affiliated Eye Hospital of Wenzhou Medical University. Participants: This study included 25 patients with proliferative diabetic retinopathy (PDR), and 25 sexand age-matched control subjects, i.e. patients with idiopathic macular hole and idiopathic epiretinal membrane. Methods: Clinical diagnoses were made by the senior ophthalmologists in the Eye Hospital; the aqueous and vitreous humour specimens were collected from these patients undergoing pars plana vitrectomy for treating complications. Main Outcome Measures: The aqueous and vitreous levels of D-serine and glutamate were measured with reverse-phase high-performance liquid chromatography (HPLC); the contents of haemoglobin in
Results: The concentrations of D-serine in the aqueous or vitreous humour were significantly higher in patients with PDR compared with control subjects. The vitreous concentrations of D-serine in PDR were 25.55 ± 0.63 μmol/L compared with control subjects at 22.76 ± 0.36 μmol/L (P = 0.002); the levels of D-serine in the aqueous humour from patients with PDR were 29.08 ± 1.31 μmol/L compared with control subjects at 24.22 ± 0.65 μmol/L (P = 0.006). Correction from possible introduction of D-serine from the vitreous haemorrhage in PDR did not significantly alter the findings. Conclusions: Increased D-serine in the aqueous and vitreous humour was found in patients with PDR compared with control subjects. Key words: diabetic retinopathy, excitotoxicity, glutamate, retinal neuronal death, serine racemase.
■ Correspondence: Dr Shengzhou Wu and Dr Zongming Song, Key Laboratory of Visual Science, National Ministry of Health, School of Optometry and Ophthalmology, Wenzhou Medical University, Zhejiang Province, China. Email: [email protected] or [email protected] Received 25 January 2014; accepted 12 March 2014. Competing/conflicts of interest: No stated conflict of interest. Funding sources: The study was supported by National Natural Science Foundation of China (81371027), by Zhejiang Province Natural Science foundation (Y2110086), by Qianjiang Scholar Scheme (QJD1202020), by Chinese Ministry of Education (20133321120002), and by start-up funding (89210001) from Wenzhou Medical University to Dr Shengzhou Wu. *Jiang Haiyan and Du Jinlin contributed to the work equally. © 2014 Royal Australian and New Zealand College of Ophthalmologists
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INTRODUCTION
Table 1.
Diabetic retinopathy (DR) is a leading cause of adult blindness and one of the most common complications of diabetes. It becomes prevalent after about a decade of diabetes, ultimately leading to retinal oedema, neovascularization and vision loss in 5% of patients.1,2 The natural history of DR has been divided into an early, non-proliferative stage, and a later, proliferative stage. The pathology of DR involves inflammation-related changes in retinal vessels and neurodegeneration in the retinal ganglion cell layer (RGCL) and inner nuclear layer (INL);3 some evidence indicate this neuronal cell death precedes vascular changes in DR.3,4 Excitotoxins including homocysteine and glutamate induce apoptosis in RGC, which suggests that excitotoxicity is associated with this RGC degeneration.5 RGC degeneration in DR linking with excitotoxicity is substantiated with a line of evidence: increased retinal/or vitreous glutamate in patients with DR and in DR animal models;6–8 altered protein levels of ionotropic glutamate receptor subunits in rat and human diabetic retina;9,10 protection of retinal neuronal loss in diabetic rats3 by memantine, an uncompetitive antagonist of N-methyl-D-aspartate receptor (NMDAR). Recently, excitotoxicity contributing to neural degeneration was also linked to activity of serine racemase (SR), an enzyme that converts L-serine to D-serine.11–14 D-serine acts as a co-agonist at the glycineB site of the NMDAR thus influences neurotransmission and fine-tunes excitotoxicity.13–15 Evidence indicate that NMDA toxicity towards RGCs is enhanced by intravitreal injection of D-serine or glycine, whereas blocking glycine transport or blocking the glycineB binding site with 5, 7dichlorokynurenic acid (DCKA) reduces toxicity.16 Our prior results demonstrate that D-serine and glutamate are increased in the aqueous humour in the STZ-induced diabetic rats.17 In this study, the levels of D-serine and glutamate in the aqueous and/or vitreous humour in patients with PDR were determined to investigate their potential roles in retina neuronal damage.
Variable
Patients’ characteristics Patients with PDR (n = 25)
Patients with IMH or IME (n = 25)
58.6 ± 9.9
65.5 ± 7.1
13 (52%) 12 (48%)
10 (40%) 15 (60%)
1 (4%) 24 (96%)
0 (0) 0 (0)
Age, mean(SD), year Sex Male Female Categories of diabetes Type 1 Type II Duration of diabetes, year 5–10 11–20 21–30 Hypertension IOP, mean (SD), mmHg Retinal detachment Vitreous haemorrhage
3 (12%) 20 (80%) 2 (8%) 12 (48%) 13.4 ± 4.0 10 (40%) 17 (68%)
0 (0) 0 (0) 0 (0) 8 (32%) 13.6 ± 2.5 0 (0) 0 (0)
IME, idiopathic epiretinal membrane; IMH, idiopathic macular hole; IOP, intraocular pressure; PDR, proliferative diabetic retinopathy; SD, standard deviation. Unless otherwise indicated, data were given as the numbers of the participating patients and as the percentages in the corresponding groups, shown in parentheses.
idiopathic macular hole and idiopathic epiretinal membrane. The undiluted specimens were provided by the surgeons in the Retinal Surgery Department of Eye Hospital in Wenzhou Medical University. The characteristics of the patients were listed in Table 1. The patients with prior vitrectomy, intraocular ischaemia, and psychiatric problems18 were excluded from the study due to considering the causes other than diabetic retinopathy. PDR was defined with the presence of neovascularization, fibroproliferation of the disc or elsewhere on the retina under fundus examination and fluorescein angiography; macular hole with the presence of small break in macular, and epiretinal membrane with the presence of abnormal membrane that grows over macular area, which were examined with optical coherence tomography (OCT). All the diagnoses were made by the senior ophthalmologists in the Retinal Surgery Department of Eye Hospital in Wenzhou Medical University.
METHODS Patients
Sample collections
The study followed the tenets of the declaration of Helsinki and was approved by the ethics committee of Wenzhou Medical University. The informed consents were obtained from the subjects after explanation of the nature and the purposes of the study. The vitreous and aqueous humour specimens were collected from patients undergoing pars plana vitrectomy, including patients with PDR and patients with
Three-port, 23-gauge pars plana vitrectomy was conducted in which vitreous cutter was introduced via transconjunctival approach. A sample of undiluted vitreous (0.4–0.6 mL) was aspirated manually through suction port into a disposable tuberculin syringe set through a cannula and a sample of undiluted aqueous (0.1–15 mL) was also collected. Fasting venous blood samples were collected for
© 2014 Royal Australian and New Zealand College of Ophthalmologists
Role of D-serine in retinal ganglion cell death glucose, haemoglobin, glycated haemoglobin, and cholesterol assays.
Materials D-serine, glutamate, glacial acetic acid, o-phthaldialdehyde (OPA), and Boc-L-Cys were obtained from Sigma (St Louis, MO, USA). All solvents of HPLC grade were purchased from Merck (Darmstadt, Germany). Ultra-pure water prepared by a Milli-Q purification system from Millipore (Bedford, MA, USA) was used for preparing the mobile phase and all other solutions.
Determination of glutamate and D-serine Reverse-phase HPLC with fluorimetric detection was used to determine glutamate, D-serine as reported before.19 Pre-column derivatization with o-phthaldialdehyde (OPA) in combination with N-tert-butyloxycarbonyl-L-cysteine was used. A 3.5-μ column (150 × 4.6 mm, ZORBAX Eclipse AAA column) was used to separate the amino acids and the methods to establish the linear gradients in mobile phase followed the previous protocol.11 The categories of samples were blinded to the researchers (Jiang HY and Du JL) conducting the analyses.
Correction for vitreous haemorrhage The correction method followed the previous procedure.6 Haemoglobin concentration in blood was determined using automatic blood cell analyser (XT1800i; Sysmex Instruments, Sysmex, Kobe, Japan) (minimum level of detection, 1.0 g/dL) and in the vitreous using spectrophotometry (722S, Shanghai Precision & Scientific Instrument Co., Ltd, Shanghai, China) (minimum level of detection, 0.03 g/dL) to correct for the possible introduction of D-serine by vitreous haemorrhage. Corrected concentrations were calculated as follows:
[ D-serine] corrected = ([ D-serine] measured × [ Hgb] blood − [D-serine] blood × [ Hgb] vitreous) ([ Hgb] blood − [ Hgb] vitreous)
843 examined with the Pearson χ2 test. P < 0.05 was used as the criterion to reject the null hypothesis.
RESULTS Our results revealed the vitreous concentrations of D-serine (25.55 ± 0.63 μmol/L) in patients with PDR were significantly higher than patients with idiopathic macular hole and idiopathic epiretinal membrane with D-serine at 22.76 ± 0.36 μmol/L (P = 0.002) (Fig. 1); the aqueous concentrations of D-serine (29.08 ± 1.31 μmol/L) in patients with PDR were also significantly higher than control subjects at 24.22 ± 0.65 μmol/L (P = 0.006). We also revealed that the concentrations of glutamate in the aqueous and vitreous humour in patients with PDR were significantly higher than those in control subjects, which is consistent with previous report that the vitreous glutamate in patients with PDR is elevated.6 Our results indicated that the vitreous glutamate in patients with PDR was 12.23 ± 1.53 μmol/L compared with control subjects at 5.65 ± 0.48 μmol/L (P = 0.021) (Fig. 1); the aqueous glutamate in patients with PDR was 15.55 ± 1.67 μmol/L compared with control subjects at 11.22 ± 0.54 μmol/L (P = 0.021). To avoid the introduction of D-serine and glutamate from the vitreous haemorrhage from PDR, we adopted the correction strategy described in the Methods. The mean blood haemoglobin concentration in patients with PDR was 121.22 ± 17.18 g/L (mean ± SD) and the vitreous haemoglobin concentration in patients with PDR was 0.49 ± 0.18 g/L (mean ± SD) determined by spectrophotometry. Corrections did not alter the results for D-serine, for instance, the vitreous level of D-serine after correction was 25.55 ± 0.635 μmol/L compared to 25.55 ± 0.63 μmol/L before correction; the aqueous level of D-serine after correction was 29.09 ± 1.32 μmol/L compared with 29.08 ± 1.31 μmol/L before corrections. Similarly, the corrections for glutamate did not significantly alter the results, for instance, the aqueous and vitreous glutamate were 11.79 ± 1.54, 15.11 ± 1.67 μmol/L, respectively, after corrections, which were significantly higher than control subjects (P < 0.001 and P = 0.053).
DISCUSSION Statistic analyses All values were given as mean ± SEM, unless otherwise indicated. Since the values of D-serine and glutamate in the aqueous or vitreous did not have normal distributions, non-parametric Mann– Whitney U-test was used to compare the difference between PDR and control subjects. The distributions of age and sex in PDR and control subjects were
We hereby indicated increased D-serine in the aqueous and vitreous humour in patients with PDR compared with the patients with idiopathic macular hole and idiopathic epiretinal membrane. To our knowledge, this is the first report to probe D-serine in the aqueous and vitreous humour in patients with PDR. Since serine racemase expression declines with ageing,20 we carefully chose the patients without age
© 2014 Royal Australian and New Zealand College of Ophthalmologists
844
Jiang et al. (b)
3 3
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(c) Fluorescent Intensity (LU)
Fluorescent Intensity (LU)
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Content of Glu and D-se (μmol/L)
Fluorescent Intensity (LU)
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D-serine
Glutamate
Figure 1. Increased D-serine and glutamate in the vitreous of patients with proliferative diabetic retinopathy (PDR), determined by high-performance liquid chromatography (HPLC). (a) Amino acid standards were separated by reverse-phase HPLC; 1: L-Asp, TR = 9.445 min; 2: L-Glu, TR = 13.45 min; 3: L-Ser, TR = 19.946 min; 4: L-Gln, TR = 20.578 min; 5: D-ser, TR = 21.752. Typical running scripts for the vitreous humour from control subjects were indicated in (b) or from patients with PDR in (c). Quantifications of D-ser and glutamate in control subjects and patients with PDR were indicated in (d). The results shown were mean ± SEM from 25 control subjects and 25 patients with PDR (*P < 0.05 vs. control).
difference between PDR and control subjects (P = 1). Simultaneously, the sex distribution in each group was also delicately designed, revealed by no difference existing between PDR and control subjects (P = 0.186). To exclude the interference from other ocular disorders, particularly with RGC death, i.e. glaucoma,21 in which RGC death may overlap the neuronal death occurring in DR, we excluded patients with high intraocular pressure (IOP) and the patients selected were without glaucoma (Table 1). Further, IOPs in patients with PDR were not different from those in control subjects (P = 0.079, Student t-test). We also collected aqueous humour from nonproliferative diabetic retinopathy patients (NPDR, n = 11) during the injections of anti-angiogenic medicines (Avastin or Lucentis) to the vitreous cavities. There were no differences on sex, age and IOP between NPDR and control subjects. The aqueous level of D-serine (32.19 ± 4.08 μmol/L), glutamate (15.66 ± 2.10 μmol/L) were significantly higher than those in control subjects (P = 0.006, 0.016, respectively). However, there were no difference in the levels of the aqueous D-serine and glutamate between patients with PDR and NPDR (P = 0.710, 0.761, respectively). In summary, our study demonstrated increased D-serine along with glutamate in the aqueous and
vitreous humour in patients with PDR, which suggests that enhanced NMDA-receptor activity is a contributing factor for RGC death occurring in DR.
ACKNOWLEDGEMENTS All the authors appreciate the dedication and patience from participants and the funding sources.
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