- Email: [email protected]
Vitreous and serum levels of transthyretin (TTR) in high myopia patients are correlated with ocular pathologies
Clinical Biochemistry 44 (2011) 681–685
Contents lists available at ScienceDirect
Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n b i o c h e m
Vitreous and serum levels of transthyretin (TTR) in high myopia patients are correlated with ocular pathologies Jun Shao a,b,1, Yu Xin c,d,1, Rongxiu Li d, Ying Fan a,⁎ a
Department of Ophthalmology, Shanghai Jiao Tong University Affiliated First People's Hospital, Haining Road 100, Shanghai 200080, People's Republic of China Department of Ophthalmology, Wuxi People's Hospital, Qing yang Road 299, Wuxi 214023, People's Republic of China School of Biotechnology, Jiang Nan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi 214036, Jiangsu, People's Republic of China d Key Laboratory of Microbial Metabolism of Ministry of Education, College of Life Science and biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's Republic of China b c
a r t i c l e
i n f o
Article history: Received 19 November 2010 Received in revised form 7 March 2011 Accepted 9 March 2011 Available online 21 March 2011 Keywords: Transthyretin High myopia Biomarker Serum Vitreous
a b s t r a c t Purpose: To detect serum and vitreous transthyretin (TTR) in high myopia patients and to evaluate potential associations between TTR and clinical parameters and ocular pathologies, including different ocular pathologies. Design and methods: Serum samples from 16 high myopia patients and 4 controls were analyzed by LTQMASS. Serum samples from 116 high myopia patients and 86 healthy controls were tested by Western blots and ELISA. Eight healthy and 40 pathologic vitreous samples were analyzed by ELISA. And corresponding serum samples were also analyzed by ELISA. Results: Significant increased TTR serum levels were detected in high myopia patients compared to healthy controls. The high levels of serum TTR were associated with ocular pathologies, long axial length, and low visual acuity. TTR in high myopia patients with macular hole and macular detachment was upregulated in both vitreous and the corresponding serum samples. TTR levels in serum samples of high myopia patients with long axial lengths were higher than in the vitreous. Conclusions: Serum TTR may be a biomarker for high myopia patients with ocular pathologies. © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction High myopia is usually defined as eyes with -6 dioptres (D) of myopia, orN26.0 mm in axial length [1]. Progressive and excessive elongation of the eyeball leads to secondary ocular diseases such as retinal detachment [2] and macular degeneration [3]. These complications can cause irreversible visual disturbances. Although the diagnosis and therapies for high myopia are improving, a high percentage of cases still result in blindness. In the Beijing Eye Study of 4409 Chinese individuals under 40 years old, high myopia was the second most frequent cause of low-vision and blindness [4]. The aging population is growing disproportionately in China, so the visual acuity of the elderly population is of great social concern [5]. To reduce the complications and improve the effectiveness of diagnosis and therapy, it is important to detect the protein biomarkers that are associated with high myopia progression as these may be useful for diagnosis or as therapeutic targets.
⁎ Corresponding author. E-mail address: [email protected] (Y. Fan). 1 These authors contributed equally to this work.
Transthyretin (TTR) is a homotetrameric protein of 55 kDa synthesized mainly in liver, choroid plexus, retinal pigment epithelium, and pancreas [6–8]. In serum, it functions as a carrier for thyroxin and retinol-binding protein (RBP). In ophthalmology research, vitreous amyloid fibrils were the result of local synthesis of mutated TTR. Nuclear cataract was associated with low protein intake and low serum levels of TTR [9]. Immunohistochemical analysis of drusen in patients with age-related macular degeneration showed the presence of TTR [10]. So far, there are few reports of TTR levels in clinical high myopia samples. Duan et al [11] reported TTR levels were elevated in the aqueous humor of five high myopia samples, but it is still uncertain if this phenotype is common in high myopia patients. In this paper, serum samples of 16 patients and 4 controls were analyzed by LTQ-MASS. We also determined TTR levels in the vitreous and corresponding serum from high myopia and normal samples by ELISA, and tested for TTR concentrations in serum samples from patients with different ocular pathologies by Western blot and ELISA. The aim of the present study was to evaluate potential associations between levels of TTR in the vitreous and serum samples of high myopia patients with clinical parameters such as age, sex, axial length, and ocular pathology.
0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.03.032
682
J. Shao et al. / Clinical Biochemistry 44 (2011) 681–685
Materials and methods
LTQ-MASS analysis
Serum samples
Total serum proteins from healthy individuals were used to immunize rabbits, and then the antibodies for human serum background proteins were coupled with CNBr-activated sepharose; serum samples from four healthy and 16 high myopia individuals were applied onto the medium, and then the 20 flow-through fractions were analyzed by LTQ-MASS. Briefly, 25 μg trypsin was dissolved in 2.5 mL tosylphenylalanylchloromethane (TCPK) mixed with 250 μL of 0.1% redistilled acetonitrile. A 15 μL sample of this trypsin solution was activated in 100 μL 50 mM NH4HCO3. A 5 μL sample from one healthy individual and the concentrated flowthrough proteins were reduced with 100 μL of reducing buffer (containing 200 μL of TCEP in 2 mL digestion buffer) and incubated at 60 °C for 10 min. Following incubation, 100 μL of alkylation buffer (containing 60 mg of iodoacetamide in 3 mL digestion buffer) was added to the tube and incubated in the dark at room temperature for 1 h for carboxymethylation and oxidation of cysteine and methionine residues. Then, 20 μL of activated trypsin solution was added to the tube, and incubated at 37 °C for 1 h and then at 25 °C overnight with gentle mixing. This peptide mixture was injected onto a Zorbax 300 SB-C18 peptide trap (Agilent Technologies, Wilmington, DE) to desalt, and separation was performed on a Zorbax 300SB-C18 reverse phase capillary column (300 μm inner diameter × 15 cm, Agilent Technologies). The mobile phases were 0.1% formic acid (A) and 84% CH3CN and 0.1% formic acid (B). The flow rate was 500 nl/min with a linear gradient of 4–50% B over 50 min, a step up to 100% B over 4 min, then 100% B for 10 min. The peak was injected online into a Finnigan LTQ (single linear quadrupole ion trap) mass spectrometer for peptide identification. Mass spectrometry was performed on a Finnigan LTQ linear ion trap. The MS method consisted of a cycle combining one full MS scan with two MS/MS events (25% collision energy). Dynamic exclusion duration was set to 30 s.
The serums of high myopia patients were obtained from ophthalmology outpatients of Shanghai Jiao Tong University Affiliated First People's Hospital. High myopia patients with axial lengths (ALs) of 26.0 mm or more were designated as study cases (n = 116), while emmetropia subjects with ALs ranging from 21.0 to 23.99 mm constituted the control cases (n = 86). Eight-six normal serum samples were obtained from healthy volunteers. Subjects with a history of intraocular surgery, ocular trauma, raised intraocular pressure, uveitis, pseudoexfoliation, diabetes mellitus, LASIK/PRK, prophylactic laser photocoagulation, and systemic diseases such as Alzheimer's disease, Parkinson's disease, schizophrenia, depression and several types of cancer, rheumatoid arthritis, glomerular disease, hepatitis, tissue injury, and inflammation were excluded from the study. The mean age of high myopia subjects was 49.7 ± 12.3 years, while the mean age of the emmetropia group was 48.5 ± 10.5 years. All study participants underwent a complete ophthalmic examination. After all the exams, patients were diagnosed with fundus manifestation of macular detachment (n = 17), macular hole (n = 14), choroidal neovascularization (n = 20), macular epimacular membrane (n = 11), atrophy (n = 28), or no significant pathology (n = 26). The mean AL of high myopia patients was 29.35 ± 2.47 mm, significantly longer (p b 0.01) than that of the normal group (24.12 ± 1.38 mm). In the high myopia group, the ALs of 48 patients ranged from 26.0 mm to 27.99 mm, the ALs of 37 patients ranged between 28 mm and 29.99 mm, and 31 patients had ALs of 30 mm or more. All patients and control subjects involved in this study were similar in social background and were from the local ethnic Han Chinese population, with no ethnic subdivision. A total amount of 5 mL of blood was collected in 10 mL glass tubes and allowed to clot for 1.5 h at room temperature. The clotted material was removed by centrifugation at 3000 rpm for 10 min. 200 μL of each serum sample was diluted with 600 μL of 20 mM PBS, separated into 4 tubes (200 μL/tube) and stored at − 20 °C for further analysis. Vitreous samples The high myopia undiluted vitreous humor samples (n = 40, 0.3 to 1.0 mL) were obtained during pars plana vitrectomy under visual control by aspirating liquefied vitreous from the center of the vitreous cavity with a syringe before the vitrectomy infusion. The corresponding serum samples were obtained before surgery. The control vitreous samples from normal human eyes with no known ocular diseases (n = 8) were obtained from eyes donated for corneal transplant (in accordance with the Standardized Rules for Development and Applications of Organ Transplants) from the Eye Bank of Shanghai in China. The normal vitreous samples (0.8 to 1.0 mL volume) were all aspirated with a syringe at pars plana. The normal serum samples were obtained from 8 healthy volunteers without any known ocular and systemic diseases. Harvested vitreous humor samples were collected in Eppendorf tubes, placed immediately on ice, centrifuged for 15 min at 12,000 rpm to separate the cellular contents; 200 μL of each sample was diluted with 600 μL of 20 mM PBS, separated into 4 tubes (200 μL/tube) and stored at − 20 °C for further analysis. Materials A TTR ELISA Kit (catalog number T771-50) was purchased from Groundwork Biotechnology Diagnosticate Ltd. (USA). A polyclonal antibody to TTR (catalog number A0002) was purchased from Dako Co. Ltd. (Denmark); other chemicals were of analytical grade and from local companies.
Western blot analysis Serum samples (1 μL) from the subjects with high myopia and normal volunteers were analyzed by Western blotting. The samples were electrophoresed on 10% SDS-PAGE gels, and then electrophoretically transferred to NC membranes (Hybond-C; Amersham Biosciences UK limited, Arlington Heights, IL) at 60 mA for 0.5 h. Membranes were blocked for 2 h at room temperature with blocking buffer (2% BSA in PBS-T) and incubated for 1–2 h at 37 °C with rabbit anti-TTR antibody (1:2,000).The membrane was washed four times for 5 min each with PBS containing 0.1% Tween-20 and then incubated with the secondary antibody (goat anti-rabbit antibody labeled with HRP) for an additional 30 min. The membrane was then washed several times and scanned using an Odyssey infrared imaging system (LI-COR, Lincoln, NE) at 700 to 800 nm. Western blots were repeated 3–5 times and qualitatively similar results were obtained each time. ELISA The concentration of TTR in serum samples from high myopia patients was determined using an enzyme linked immunosorbent assay (ELISA, Groundwork Biotechnology Diagnosticate Ltd.) following the manufacturer's instructions. and then the samples (50 μL) were added into wells pre-coated with antibody, and 100 μL of 5 % BSA was added to each well. The wells were covered and incubated for 1 h at 37 °C. All wells were then washed five times with distilled or deionized water. The HRP-coupled antibody was added and the wells were recovered and incubated for 1 h at 37 °C. All wells were then washed five times with distilled or de-ionized water. Then, 50 μL of substrate (0.1 % tetramethylbenzidine) was added to each well. The wells were covered and incubated for 15 min at 20–25 °C, followed by
J. Shao et al. / Clinical Biochemistry 44 (2011) 681–685
addition of 50 μL stop solution (2 M H2SO4). The OD450nm was measured on a microplate reader within 30 min. Statistical analysis All values are expressed as mean ± SEM. Statistical analysis based on Student's t-tests were scored to identify significant differences in multiple comparisons (SPSS 11.5, Chicago, IL). A level of p b 0.05 was considered statistically significant. Ethics The research followed the tenets of the Declaration of Helsinki for the use of human subjects. Informed consents were obtained from all subjects after verbal and written explanation of the nature and possible consequences of the study. The ethics committee of the Shanghai Jiao Tong University approved the research protocol. Results LTQ-MASS analysis After reduction and carboxymethylation of cysteine using iodoacetamide, all the flow-through proteins were hydrolyzed into peptides by trypsin, desalted on Zorbax 300 SB-C18 peptide traps (Agilent Technologies, Wilmington, DE), separated on a Zorbax 300SB-C18 reverse phase capillary column, and subjected to LTQMASS analysis; two healthy serum samples were used as controls without the subtractive protocol. The MS/MS spectra acquired from all the runs were searched against the IPI HUMAN v3.36 database using the program SEQUEST. The SEQUEST filter was set to Xcorr ≥ 1.9 for Charge +1, Xcorr ≥ 2.2 for Charge + 2, Xcorr ≥ 3.75 for Charge + 3, and DelCN ≥ 0.1. In the flow-through fractions of 4 healthy serum samples, peptides of 25, 34, 18 and 25 different proteins were identified. Several highly abundant proteins, such as albumin, serotransferrin, and fibrinogen were identified by N 3 unique peptides, while most of the background proteins were removed. In the flow-through fractions of 16 serum samples from hereditary high myopia, peptides from 57, 32, 54, 63, 88, 70, 122, 108, 20, 43, 25, 18, 19, 24, 68 and 146 proteins were identified. Compared to serum samples from healthy individuals, a number of unique proteins were identified (Table 1). Transthyretin (TTR) was the most significant of these unique proteins in high myopia samples. Indeed, it was identified in the flow-through fractions of all 16 abnormal serum samples, while no TTR was identified in any sample from healthy individuals. Western-blot analysis of TTR in serum samples The different levels of TTR in serum samples from health and high myopia patients were confirmed by Western blots. Levels of TTR in samples from patients with ALs greater than 26 mm were higher than
683
from patients with ALs below 26 mm. Increased TTR band intensity in high myopia groups was correlated with increasing AL. Samples from high myopia patients with ocular pathology had higher TTR concentrations than samples from healthy controls or samples from high myopia patients with no significant ocular pathology (Fig. 1). The relation between TTR and clinical profile in high myopia patients The clinical features of high myopia patients, including visual acuity, axial length, various ocular pathologies age and sex are listed in Tables 2 and 3. The serum TTR in normal controls did not show any significant association with sex, age (p N 0.05); however, TTR levels of serum samples in high myopia patients was correlated with ocular pathological (p b 0.01) and axial length (p b 0.01). The average TTR concentration of serum samples from macular detachment patients (235.5 ± 23.0 mg/L), macular hole patients (131.9 ± 15.0 mg/L) and choroidal neovascularization patients (94.0 ± 6.0 mg/L) was significantly higher than the control samples (32.3 ± 8.8 mg/L). In addition, the average TTR concentration in serum samples from patients with axial lengths of 30 mm or more (182.0 ± 28.0 mg/L), from 26.5 mm to 27.99 mm (60.5 ± 9.5 mg/L), and from 28 mm to 29.99 mm (98.0 ± 15.0 mg/L) were all significantly higher than in samples from healthy individuals (32.3 ± 8.8 mg/L). TTR levels in vitreous and corresponding serum samples It was noteworthy that TTR could be detected in all vitreous samples and the corresponding serum samples. The TTR concentration of high myopia patients with macular detachment and macular hole was significantly higher in both vitreous and corresponding serum samples. However, the TTR concentration in vitreous samples from patients with long axis lengths was lower than in the corresponding serum samples (p N 0.05) (Table 4). Visual acuity was well correlated with vitreous TTR. We found that patients with better visual acuity post-operatively had higher TTR concentrations in vitreous and lower concentration in serum (Table 5). Thus, TTR may be a promising candidate serum biomarker for the evaluation of different ocular pathologies and visual acuity. Discussion We found that TTR levels were higher in serum samples from patients with high myopia, especial in patients with ocular pathologies. The TTR concentration of serum samples in the high myopia patients with macula detachment, macular hole, choroidal neovascularization, macular epimacular membrane, and atrophy (235.5 ± 23.0 mg/L, 131.9 ± 15.0 mg/L, 94.0 ± 6.0 mg/L, 84.1 ± 2.0 mg/L, and 70.6 ± 3.5 mg/L respectively) were dramatically higher than samples from healthy patients (32.3 ± 8.8 mg/L). These results underscore the high correlation between elevated serum TTR and high myopia patients with ocular pathology. Studies on the pathogenesis of high myopia indicate that genetic influences played a significant role in its
Table 1 The significantly differential proteins in hereditary high myopia patients' serums. Differential proteins
Protein occurrence frequency in 4 healthy serums' flow-through fractions
Protein occurrence frequency Description in 16 abnormal serums' flow-through fractions
Hemopexin Apo B-100 Transthyretin (TTR)
1 1 0
16 12 16
Haptoglobin (HP)/Haptoglobin-related 0 protein (HPr)
16
The relationship with high myopia is unclear. Associated with age-related maculopathy Associated with vitreous amyloidosis, nerve amyloid, ocular amyloid angiopathy, and other ocular related disease. Associated with self immunization; higher serum haptoglobin levels in patients with Behcet's disease (an ocular related disease) compared to control subjects were obtained.
684
J. Shao et al. / Clinical Biochemistry 44 (2011) 681–685
Fig 1. The levels of TTR were detected in serum of normal individuals and high myopia patients by western blot. A: 1, 2, 9, 10 represented TTR levels of macular hole patients in serum; 3, 4, 11, 12 represented TTR levels of central fovea of macula detachment patients in serum; 5, 6 represented TTR levels of atrophy patients in serum; 7, 8 represented TTR levels of normal individuals in serum. 13, 14 represented TTR levels of choroidal neovascularization patients, 15 represented TTR levels of macular epimacular membrane patients in serum, 16 represented TTR levels of patients with no significant ocular pathology in serum. B: 1, 2 represented TTR levels of AL was 30 mm or more ; 3, 4 represented TTR levels of AL ranging from 28 mm to 29.99 mm; 5, 6 represented TTR levels of AL ranging from 26.0 mm to 27.99 mm ; 7, 8,13,14,15,16 represented TTR levels of normal individuals in serum. 9–12 represented TTR levels of high myopia patients in serum. In conclusion, TTR level in serum increased accompany by growth of AL; each pathologic appearance of TTR level in serum was significantly different.
development [12]; however, genetic analysis provided an incomplete picture. Proteomic analysis can provide a more detailed picture of the relationship between gene sequences and cellular physiology [13], and so can complement genetic analysis for evaluating disease development, prognosis, and response to treatment [14]. Our LQTMASS results demonstrate the value of identifying and detecting protein biomarkers for disease, in this case for high myopia, that can be employed for diagnosis and monitoring. Consistent with the findings of van Aken et al. [15], our results also demonstrated that visual acuity was positively correlated with vitreous TTR concentrations. The average vitreous TTR concentration of patients with visual acuity N 0.1 (140.5 ± 4.5 mg/L) was significantly higher than in patients with visual acuity b 0.1 (89.5 ± 5.5 mg/L). A higher metabolic rate in cells of the retinal pigment epithelium (RPE) in high myopia patients with better visual acuity may account for this higher TTR levels. The RPE of the eye and choroid plexus of the brain are developmental and functional homologues, sharing many of the characteristics of transporting epithelia; cells are joined by tight apical membrane junctions and they regulate the transport of fluids and serum proteins into their respective humors. The expression of TTR mRNA in RPE cells is higher than all other tissues except for the choroid plexus [16]. The TTR protein is secreted predominantly in an apical direction by RPE cells in vitro [17]. The source of all-transretinol for the RPE is the liver, which secretes retinol bound to the TTR–RBP complex. In RPE cells, free retinol uptake appears to be mediated by STRA6, a retinol-RBP cell membrane receptor that is highly expressed on the basolateral side of these cells [18]. The physiological role of TTR in the eye is not completely known, but it may participate in retinol cycling. The fact that retinol metabolism
Table 2 TTR concentrations of high myopia patients’ serum samples. Clinical parameter Visual acuity
Axial length
Pathological appearance
N 0.5 0.1–0.5 b 0.1 N 30 28–30 26–27.99 macular detachment macular hole choroidal neovascularization macular epimacular membrane Atrophy no significant pathological changes
was altered in TTR-null mice suggests that TTR plays a major role in retinol metabolism in the interphotoreceptor matrix (IPM) [19]. Almost all eye diseases are coupled with destruction of bloodretina barriers. It was reported that high myopia patients with ocular pathologies showed accompanying changes in blood-retinal barrier permeability [20]. In the present report, the mean TTR concentrations of serum samples in patients with macular hole and macular detachment (109.3 ± 24.5 mg/L, 165.5 ± 25.5 mg/L) were similar to the vitreous concentrations (99.2 ± 23.5 mg/L, 121.9 ± 25.5 mg/L), but still much higher than the average TTR concentration from vitreous samples from healthy subjects (44.5 ± 7.5 mg/L). Macular hole and macular detachment are thought to be caused by traction of the vitreous, long axial length, posterior staphyloma, and RPE atrophy. In response to these kinds of damage, TTR in the vitreous might flow into the serum from cells and tissues of the eye through damaged blood-retina barriers. To maintain vitreous TTR, high rates of synthesis might be required, thus explaining the high vitreous TTR concentrations in these conditions. Furthermore, the high serum TTR concentrations may be associated with destruction of blood–retina barriers. Retinal and choroid atrophy are usually caused by continuous extension of the sclera posterior pole, and the RPE denaturizing for retinal and choroidal vascular distortion [21]. Retinal scars and choroid atrophy are often detected in the development of late high myopia [22]. Irreversible RPE damage should lower serum TTR concentrations. However, the serum TTR concentration of patients with macular epimacular membrane was higher than samples from health individuals because of increased TTR secretion caused by proliferation of RPE cells and Müller glial [23]. Additionally, the increased level of TTR in the retinal blood circle could stimulate the proliferation of RPE. The TTR concentration of serum samples in high myopia patients with choroidal neovascularization (94.0 ± 6.0 mg/L) was upregulated. It was reported that estrogens and androgens up-regulate TTR gene expression in liver and choroid plexus in mammals [24]. Estrogen receptors were also found in bovine retina and especially on vessels in the choroid [25,26]. Recently, several studies demonstrated the expression of estrogen receptors in human retina and retinal pigment epithelium [27,28]. Estrogen receptors were reported to be expressed in the CNV of highly myopic eyes [29]. Transthyretin might stimulate estrogen receptors to increase TTR synthesis. The average TTR
Number of patients
TTR concentration
p Value
28 50 38 31 37 48 17 14 20
112.4 ± 36.5 mg/L 97.6 ± 44.5 mg/L 108.7 ± 34.0 mg/L 182.0 ± 28.0 mg/L 98.0 ± 15.0 mg/L 60.5 ± 9.5 mg/L 235.5 ± 23.0 mg/L 131.9 ± 15.0 mg/L 94.0 ± 6.0 mg/L
N 0.05
11
84.1 ± 2.0 mg/L
Age
28 26
70.6 ± 3.5 mg/L 55.0 ± 3.5 mg/L
Sex
b 0.01
b 0.01
Table 3 TTR concentrations in controls' serum samples. Age/sex b 40 40 ~ 50 N 50 female male
Number of controls
TTR concentration
p Value
20 56 10 44 42
27.8 ± 3.2 mg/L 35.9 ± 9.3 mg/L 21.1 ± 5.8 mg/mL 33.9 ± 5.3 mg/L 31.6 ± 4.7 mg/L
N 0.05
N 0.05
J. Shao et al. / Clinical Biochemistry 44 (2011) 681–685
References
Table 4 TTR concentration in serum and vitreous samples. Clinical parameter
Number of Concentration of TTR in patients different samples
Pathological 40 appearance Macular detachment 18 Macular hole 22 None 8 Axial length N 30 28–30 26–27.99 b 26
18 12 10 8
vitreous
685
p Value
serum
121.9 ± 25.5 mg/L 165.5 ± 25.5 mg/L N 0.05 99.2 ± 23.5 mg/L 109.3 ± 24.5 mg/L N 0.05 44.5 ± 7.5 mg/L 33.0 ± 9.0 mg/L N 0.05
129.8 ± 12.0 mg/L 188.5 ± 15.5 mg/L 96.0 ± 7.0 mg/L 115.0 ± 6.0 mg/L 88.5 ± 12.0 mg/L 60.5 ± 3.5 mg/L 45.6 ± 5.6 mg/L 33.5 ± 4.3 mg/L
N 0.05 N 0.05 N 0.05 N 0.05
Table 5 The association between visual acuity postoperatively and TTR concentration in serum and vitreous samples. Visual acuity postoperatively
Number of patients
Vitreous
Serum
N 0.5 0.1–0.5 b 0.1 p
0 15 25
0 140.5 ± 4.5 mg/L 89.5 ± 5.5 mg/L p b 0.05
0 96.4 ± 12.7 mg/L 157.8 ± 11.5 mg/L p b 0.05
concentration of serum samples in patients with no significant ocular pathologies (55.0 ± 3.5 mg/L) was lower than in high myopia cases with ocular pathologies, suggesting that TTR might be a promising candidate serum biomarker for evaluation the risk for ocular pathologies in high myopia patients. TTR has been associated with a number of pathological conditions, including Alzheimer's disease, Parkinson's disease, schizophrenia, and depression. Other reports suggest that TTR was associated several types of cancer, diabetes, rheumatoid arthritis, glomerular disease, and hepatitis. Moreover, TTR is thought to be directly and causally involved in the establishment of all stages of the stress response to nutritional impairment and in acute stressful conditions, such as tissue injury. Thus, TTR is also as an indicator of malnutrition. In our study, we eliminated these factors by the strict selection of subjects. TTR is an age-associated protein [30]. We discriminated the effect of age and of clinical symptoms of TTR levels by analyzing TTR concentration of normal group. In normal group, our experiment showed that TTR levels of the serum of subjects younger than 40 years old, 40–50 years old, and over 50 years old were 27.8 ±3.2 mg/L, 35.9± 9.3 mg/L and 21.1 ± 5.8 mg/mL, respectively, there were no differences in physical conditions within the different age group. So it indicated that age is not a significant factor influencing the difference in serum TTR concentrations between ages and clinical symptoms. We showed that TTR concentrations in the vitreous samples (129.8 ± 12.0 mg/L) of patients with long AL (N 30 mm) were lower than in the corresponding serum samples (188.5 ± 15.5 mg/L). High myopia patients with long axial length had poor visual function [31], indicating that low TTR may be used as a biochemical marker for assessing visual disturbances. It is noteworthy that TTR may be one of the candidate serum biomarkers of high myopia. However, confirmation still requires large-scale studies. Further research is also needed to examine the role of TTR in the pathogenesis of ocular diseases and to develop new methods for the prevention and control of high myopia. Acknowledgments This work was supported by the program of the Key Laboratory of fundus oculi diseases, Shanghai, China (No. YDB-0901).
[1] Grossniklaus HE, Green WR. Pathologic findings in pathologic myopia. Retina 1992;12:127–33. [2] Lam CS, Goldschmidt E, Edwards MH. Prevalence of myopia in local and international schools in Hong Kong. Optom Vis Sci 2004;81:317–22. [3] Avila MP, Weiter JJ, Jalkh AE, Trempe CL, Pruett RC. Natural history of choroidal neovascularization in degenerative myopia. Ophthalmology 1984;91:1573–81. [4] Xu L, Wang Y, Li Y, Wang Y, Cui T, Li J, Jonas JB. Causes of blindness and visual impairment in urban and rural areas in Beijing. The Beijing Eye Study. Ophthalmology 2006;113:1134–41. [5] Wong TY, Foster PJ, Hee J, Ng TP, Tielsch JM, Chew SJ, Johnson GJ, Seah SK. Prevalence and risk factors for refractive errors in adult Chinese in Singapore. Invest Ophthalmol Vis Sci 2000;41:2486–94. [6] Herbert J, Wilcox JN, Pham KC, Fremeau RT, Zaviani M, Dwork A, Soprano D, Makover A, Goodman DS, Zimmerman EZ, Roberts JL, Schon EA. Transthyretin: a choroid plexus specific transport protein in human brain. Neurology 1986;36:900–11. [7] Jacobsson B, Collins VP, Grimelius L, Pettersson T, Sandstedt B. Carlstrom A Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic waslets. J chem Cytochem 1989;37:31–7. [8] Monaco HL, Rizzi M, Coda A. Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 1995;268:1039–41. [9] Delcourt C, Dupuy AM, Carriere I, Lacroux A, Cristol JP. Albumin and transthyretin as risk factors for cataract: the POLA study. Arch Ophthalmol 2005;123:225–32. [10] Mullins RF, Russell SR, Anderson DH, Hageman GS. Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. FASEB J 2000;14:835–46. [11] Duan X, Lu Q, Xue P, Zhang H, Dong Z, Yang F, Wang N. Proteomic analysis of aqueous humor from patients with myopia. Mol Vis 2008;14:370–3. [12] Jacobia FK, Zrennera E, Broghammerb M, Puschb CM. A genetic perspective on myopia cell. Mol Life Sci 2005;62:800–8. [13] Young TL, Metlapally R, Shay A. Complex trait genetics of refractive error. Arch Ophthalmol 2007;125:38–48. [14] Dove A. Proteomics: translating genomics into products? Nat Biotechnol 1999;17: 233–6. [15] Van Aken E, De Letter EA, Veckeneer M, Derycke L, van Enschot T, Geers I, Delanghe S, Delanghe JR. Transthyretin levels in the vitreous correlate with change in visual acuity after vitrectomy. Br J Ophthalmol 2009;93:1539–45. [16] Pfeffer BA, Becerra SP, Borst DE, et al. Expression of transthyretin and retinol binding protein mRNAs and secretion of transthyretin by cultured monkey retinal pigment epithelium. Mol Vis 2004;10:23–30. [17] Ong DE, Davis JT, O'Day WT, et al. Synthesis and secretion of retinol-binding protein and transthyretin by cultured retinal pigment epithelium. Biochemistry 1994;33:1835–42. [18] Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, Wiita P, Bok D, Sun H. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 2007;315:820–5. [19] Palha JA, Nissanov J, Fernandes R, Sousa JC, Bertrand L, Dratman MB. Thyroid hormone distribution in the mouse brain: the role of transthyretin. Neuroscience 2002;113:837–47. [20] Kitaya N, Ishiko S, Abiko T, Mori F, Kagokawa H, Kojima M, Saito K, Yoshida A. Changes in blood–retinal barrier permeability in form deprivation myopia in tree shrews. Vis Res 2000;40:2369–77. [21] Miller DG, Singerman LJ. Natural history of choroidal neovascularization in high myopia. Curr Opin Ophthalmol 2001;12:222–4. [22] Ohno-Matsui K, Yoshida T, Futagami S, Yasuzumi K, Shimada N, Kojima A, Tokoro T, Mochizuki M. Patchy atrophy and lacquer cracks predispose to the development of high myopia of choroidal neovascularisation in pathological myopia. Br J Ophthalmol 2003;87:570–3. [23] Garweg JG, Bergstein D, Windisch B, Koerner F, Halberstadt M. Recovery of visual field and acuity after removal of epiretinal and inner limiting membranes. Br J Ophthalmol 2008;92(2):220–4. [24] Tang YP, Haslam SZ, Conrad SE, Sisk CL. Estrogen increases brain expression of the mRNA encoding transthyretin, an amyloid beta scavenger protein. J Alzheimers Dis 2004;6:413–20. [25] Goncalves I, Alves CH, Quintela T, Baltazar G, Socorro S, Saraiva MJ, Abreu R, Santos CR. Transthyretin was up-regulated by sex hormones in mice liver. Mol Cell Biochem 2008;317:137–42. [26] Kobayashi K, Kobayashi H, Ueda M, Honda Y. Estrogen receptor expression in bovine and rat retina. Invest Ophthalmol Vis Sci 1998;39:2105–10. [27] Ogueta SB, Schwartz SD, Yamashita CK, Farber DB. Estrogen receptor in the human eye: influence of gender and age on gene expression. Invest Ophthalmol Vis Sci 1999;40:1906–11. [28] Wickham LA, Gao J, Toda I, Rocha EM, Ono M, Sullivan DA. Identification of androgen, estrogen and progesterone receptor mRNAs in the eye. Acta Ophthalmol Scand 2000;78:146–53. [29] Kobayashi K, Mandai M, Suzuma I, Kobayashi H, Okinami S. Expression of estrogen receptor in the choroidal neovascular membrane in highly myopic eyes. Retina 2002;22:418–22. [30] Ando Yukio, Nakamura Masaaki, Araki Shukuro. Transthyretin-related familial amyloidotic polyneuropathy. Arch Neurol 2005;62:1057–62. [31] Nakanishi H, Kuriyama S, Saito I, Okada M, Kita M, Kurimoto Y, Kimura H, Takagi H, Yoshimura N. Prognostic factor analysis in pars plana vitrectomy for retinal detachment attributable to macular hole in high myopia: a multicenter study. Am J Ophthalmol 2008;146(2):198–204.
Contents lists available at ScienceDirect
Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n b i o c h e m
Vitreous and serum levels of transthyretin (TTR) in high myopia patients are correlated with ocular pathologies Jun Shao a,b,1, Yu Xin c,d,1, Rongxiu Li d, Ying Fan a,⁎ a
Department of Ophthalmology, Shanghai Jiao Tong University Affiliated First People's Hospital, Haining Road 100, Shanghai 200080, People's Republic of China Department of Ophthalmology, Wuxi People's Hospital, Qing yang Road 299, Wuxi 214023, People's Republic of China School of Biotechnology, Jiang Nan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi 214036, Jiangsu, People's Republic of China d Key Laboratory of Microbial Metabolism of Ministry of Education, College of Life Science and biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's Republic of China b c
a r t i c l e
i n f o
Article history: Received 19 November 2010 Received in revised form 7 March 2011 Accepted 9 March 2011 Available online 21 March 2011 Keywords: Transthyretin High myopia Biomarker Serum Vitreous
a b s t r a c t Purpose: To detect serum and vitreous transthyretin (TTR) in high myopia patients and to evaluate potential associations between TTR and clinical parameters and ocular pathologies, including different ocular pathologies. Design and methods: Serum samples from 16 high myopia patients and 4 controls were analyzed by LTQMASS. Serum samples from 116 high myopia patients and 86 healthy controls were tested by Western blots and ELISA. Eight healthy and 40 pathologic vitreous samples were analyzed by ELISA. And corresponding serum samples were also analyzed by ELISA. Results: Significant increased TTR serum levels were detected in high myopia patients compared to healthy controls. The high levels of serum TTR were associated with ocular pathologies, long axial length, and low visual acuity. TTR in high myopia patients with macular hole and macular detachment was upregulated in both vitreous and the corresponding serum samples. TTR levels in serum samples of high myopia patients with long axial lengths were higher than in the vitreous. Conclusions: Serum TTR may be a biomarker for high myopia patients with ocular pathologies. © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction High myopia is usually defined as eyes with -6 dioptres (D) of myopia, orN26.0 mm in axial length [1]. Progressive and excessive elongation of the eyeball leads to secondary ocular diseases such as retinal detachment [2] and macular degeneration [3]. These complications can cause irreversible visual disturbances. Although the diagnosis and therapies for high myopia are improving, a high percentage of cases still result in blindness. In the Beijing Eye Study of 4409 Chinese individuals under 40 years old, high myopia was the second most frequent cause of low-vision and blindness [4]. The aging population is growing disproportionately in China, so the visual acuity of the elderly population is of great social concern [5]. To reduce the complications and improve the effectiveness of diagnosis and therapy, it is important to detect the protein biomarkers that are associated with high myopia progression as these may be useful for diagnosis or as therapeutic targets.
⁎ Corresponding author. E-mail address: [email protected] (Y. Fan). 1 These authors contributed equally to this work.
Transthyretin (TTR) is a homotetrameric protein of 55 kDa synthesized mainly in liver, choroid plexus, retinal pigment epithelium, and pancreas [6–8]. In serum, it functions as a carrier for thyroxin and retinol-binding protein (RBP). In ophthalmology research, vitreous amyloid fibrils were the result of local synthesis of mutated TTR. Nuclear cataract was associated with low protein intake and low serum levels of TTR [9]. Immunohistochemical analysis of drusen in patients with age-related macular degeneration showed the presence of TTR [10]. So far, there are few reports of TTR levels in clinical high myopia samples. Duan et al [11] reported TTR levels were elevated in the aqueous humor of five high myopia samples, but it is still uncertain if this phenotype is common in high myopia patients. In this paper, serum samples of 16 patients and 4 controls were analyzed by LTQ-MASS. We also determined TTR levels in the vitreous and corresponding serum from high myopia and normal samples by ELISA, and tested for TTR concentrations in serum samples from patients with different ocular pathologies by Western blot and ELISA. The aim of the present study was to evaluate potential associations between levels of TTR in the vitreous and serum samples of high myopia patients with clinical parameters such as age, sex, axial length, and ocular pathology.
0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.03.032
682
J. Shao et al. / Clinical Biochemistry 44 (2011) 681–685
Materials and methods
LTQ-MASS analysis
Serum samples
Total serum proteins from healthy individuals were used to immunize rabbits, and then the antibodies for human serum background proteins were coupled with CNBr-activated sepharose; serum samples from four healthy and 16 high myopia individuals were applied onto the medium, and then the 20 flow-through fractions were analyzed by LTQ-MASS. Briefly, 25 μg trypsin was dissolved in 2.5 mL tosylphenylalanylchloromethane (TCPK) mixed with 250 μL of 0.1% redistilled acetonitrile. A 15 μL sample of this trypsin solution was activated in 100 μL 50 mM NH4HCO3. A 5 μL sample from one healthy individual and the concentrated flowthrough proteins were reduced with 100 μL of reducing buffer (containing 200 μL of TCEP in 2 mL digestion buffer) and incubated at 60 °C for 10 min. Following incubation, 100 μL of alkylation buffer (containing 60 mg of iodoacetamide in 3 mL digestion buffer) was added to the tube and incubated in the dark at room temperature for 1 h for carboxymethylation and oxidation of cysteine and methionine residues. Then, 20 μL of activated trypsin solution was added to the tube, and incubated at 37 °C for 1 h and then at 25 °C overnight with gentle mixing. This peptide mixture was injected onto a Zorbax 300 SB-C18 peptide trap (Agilent Technologies, Wilmington, DE) to desalt, and separation was performed on a Zorbax 300SB-C18 reverse phase capillary column (300 μm inner diameter × 15 cm, Agilent Technologies). The mobile phases were 0.1% formic acid (A) and 84% CH3CN and 0.1% formic acid (B). The flow rate was 500 nl/min with a linear gradient of 4–50% B over 50 min, a step up to 100% B over 4 min, then 100% B for 10 min. The peak was injected online into a Finnigan LTQ (single linear quadrupole ion trap) mass spectrometer for peptide identification. Mass spectrometry was performed on a Finnigan LTQ linear ion trap. The MS method consisted of a cycle combining one full MS scan with two MS/MS events (25% collision energy). Dynamic exclusion duration was set to 30 s.
The serums of high myopia patients were obtained from ophthalmology outpatients of Shanghai Jiao Tong University Affiliated First People's Hospital. High myopia patients with axial lengths (ALs) of 26.0 mm or more were designated as study cases (n = 116), while emmetropia subjects with ALs ranging from 21.0 to 23.99 mm constituted the control cases (n = 86). Eight-six normal serum samples were obtained from healthy volunteers. Subjects with a history of intraocular surgery, ocular trauma, raised intraocular pressure, uveitis, pseudoexfoliation, diabetes mellitus, LASIK/PRK, prophylactic laser photocoagulation, and systemic diseases such as Alzheimer's disease, Parkinson's disease, schizophrenia, depression and several types of cancer, rheumatoid arthritis, glomerular disease, hepatitis, tissue injury, and inflammation were excluded from the study. The mean age of high myopia subjects was 49.7 ± 12.3 years, while the mean age of the emmetropia group was 48.5 ± 10.5 years. All study participants underwent a complete ophthalmic examination. After all the exams, patients were diagnosed with fundus manifestation of macular detachment (n = 17), macular hole (n = 14), choroidal neovascularization (n = 20), macular epimacular membrane (n = 11), atrophy (n = 28), or no significant pathology (n = 26). The mean AL of high myopia patients was 29.35 ± 2.47 mm, significantly longer (p b 0.01) than that of the normal group (24.12 ± 1.38 mm). In the high myopia group, the ALs of 48 patients ranged from 26.0 mm to 27.99 mm, the ALs of 37 patients ranged between 28 mm and 29.99 mm, and 31 patients had ALs of 30 mm or more. All patients and control subjects involved in this study were similar in social background and were from the local ethnic Han Chinese population, with no ethnic subdivision. A total amount of 5 mL of blood was collected in 10 mL glass tubes and allowed to clot for 1.5 h at room temperature. The clotted material was removed by centrifugation at 3000 rpm for 10 min. 200 μL of each serum sample was diluted with 600 μL of 20 mM PBS, separated into 4 tubes (200 μL/tube) and stored at − 20 °C for further analysis. Vitreous samples The high myopia undiluted vitreous humor samples (n = 40, 0.3 to 1.0 mL) were obtained during pars plana vitrectomy under visual control by aspirating liquefied vitreous from the center of the vitreous cavity with a syringe before the vitrectomy infusion. The corresponding serum samples were obtained before surgery. The control vitreous samples from normal human eyes with no known ocular diseases (n = 8) were obtained from eyes donated for corneal transplant (in accordance with the Standardized Rules for Development and Applications of Organ Transplants) from the Eye Bank of Shanghai in China. The normal vitreous samples (0.8 to 1.0 mL volume) were all aspirated with a syringe at pars plana. The normal serum samples were obtained from 8 healthy volunteers without any known ocular and systemic diseases. Harvested vitreous humor samples were collected in Eppendorf tubes, placed immediately on ice, centrifuged for 15 min at 12,000 rpm to separate the cellular contents; 200 μL of each sample was diluted with 600 μL of 20 mM PBS, separated into 4 tubes (200 μL/tube) and stored at − 20 °C for further analysis. Materials A TTR ELISA Kit (catalog number T771-50) was purchased from Groundwork Biotechnology Diagnosticate Ltd. (USA). A polyclonal antibody to TTR (catalog number A0002) was purchased from Dako Co. Ltd. (Denmark); other chemicals were of analytical grade and from local companies.
Western blot analysis Serum samples (1 μL) from the subjects with high myopia and normal volunteers were analyzed by Western blotting. The samples were electrophoresed on 10% SDS-PAGE gels, and then electrophoretically transferred to NC membranes (Hybond-C; Amersham Biosciences UK limited, Arlington Heights, IL) at 60 mA for 0.5 h. Membranes were blocked for 2 h at room temperature with blocking buffer (2% BSA in PBS-T) and incubated for 1–2 h at 37 °C with rabbit anti-TTR antibody (1:2,000).The membrane was washed four times for 5 min each with PBS containing 0.1% Tween-20 and then incubated with the secondary antibody (goat anti-rabbit antibody labeled with HRP) for an additional 30 min. The membrane was then washed several times and scanned using an Odyssey infrared imaging system (LI-COR, Lincoln, NE) at 700 to 800 nm. Western blots were repeated 3–5 times and qualitatively similar results were obtained each time. ELISA The concentration of TTR in serum samples from high myopia patients was determined using an enzyme linked immunosorbent assay (ELISA, Groundwork Biotechnology Diagnosticate Ltd.) following the manufacturer's instructions. and then the samples (50 μL) were added into wells pre-coated with antibody, and 100 μL of 5 % BSA was added to each well. The wells were covered and incubated for 1 h at 37 °C. All wells were then washed five times with distilled or deionized water. The HRP-coupled antibody was added and the wells were recovered and incubated for 1 h at 37 °C. All wells were then washed five times with distilled or de-ionized water. Then, 50 μL of substrate (0.1 % tetramethylbenzidine) was added to each well. The wells were covered and incubated for 15 min at 20–25 °C, followed by
J. Shao et al. / Clinical Biochemistry 44 (2011) 681–685
addition of 50 μL stop solution (2 M H2SO4). The OD450nm was measured on a microplate reader within 30 min. Statistical analysis All values are expressed as mean ± SEM. Statistical analysis based on Student's t-tests were scored to identify significant differences in multiple comparisons (SPSS 11.5, Chicago, IL). A level of p b 0.05 was considered statistically significant. Ethics The research followed the tenets of the Declaration of Helsinki for the use of human subjects. Informed consents were obtained from all subjects after verbal and written explanation of the nature and possible consequences of the study. The ethics committee of the Shanghai Jiao Tong University approved the research protocol. Results LTQ-MASS analysis After reduction and carboxymethylation of cysteine using iodoacetamide, all the flow-through proteins were hydrolyzed into peptides by trypsin, desalted on Zorbax 300 SB-C18 peptide traps (Agilent Technologies, Wilmington, DE), separated on a Zorbax 300SB-C18 reverse phase capillary column, and subjected to LTQMASS analysis; two healthy serum samples were used as controls without the subtractive protocol. The MS/MS spectra acquired from all the runs were searched against the IPI HUMAN v3.36 database using the program SEQUEST. The SEQUEST filter was set to Xcorr ≥ 1.9 for Charge +1, Xcorr ≥ 2.2 for Charge + 2, Xcorr ≥ 3.75 for Charge + 3, and DelCN ≥ 0.1. In the flow-through fractions of 4 healthy serum samples, peptides of 25, 34, 18 and 25 different proteins were identified. Several highly abundant proteins, such as albumin, serotransferrin, and fibrinogen were identified by N 3 unique peptides, while most of the background proteins were removed. In the flow-through fractions of 16 serum samples from hereditary high myopia, peptides from 57, 32, 54, 63, 88, 70, 122, 108, 20, 43, 25, 18, 19, 24, 68 and 146 proteins were identified. Compared to serum samples from healthy individuals, a number of unique proteins were identified (Table 1). Transthyretin (TTR) was the most significant of these unique proteins in high myopia samples. Indeed, it was identified in the flow-through fractions of all 16 abnormal serum samples, while no TTR was identified in any sample from healthy individuals. Western-blot analysis of TTR in serum samples The different levels of TTR in serum samples from health and high myopia patients were confirmed by Western blots. Levels of TTR in samples from patients with ALs greater than 26 mm were higher than
683
from patients with ALs below 26 mm. Increased TTR band intensity in high myopia groups was correlated with increasing AL. Samples from high myopia patients with ocular pathology had higher TTR concentrations than samples from healthy controls or samples from high myopia patients with no significant ocular pathology (Fig. 1). The relation between TTR and clinical profile in high myopia patients The clinical features of high myopia patients, including visual acuity, axial length, various ocular pathologies age and sex are listed in Tables 2 and 3. The serum TTR in normal controls did not show any significant association with sex, age (p N 0.05); however, TTR levels of serum samples in high myopia patients was correlated with ocular pathological (p b 0.01) and axial length (p b 0.01). The average TTR concentration of serum samples from macular detachment patients (235.5 ± 23.0 mg/L), macular hole patients (131.9 ± 15.0 mg/L) and choroidal neovascularization patients (94.0 ± 6.0 mg/L) was significantly higher than the control samples (32.3 ± 8.8 mg/L). In addition, the average TTR concentration in serum samples from patients with axial lengths of 30 mm or more (182.0 ± 28.0 mg/L), from 26.5 mm to 27.99 mm (60.5 ± 9.5 mg/L), and from 28 mm to 29.99 mm (98.0 ± 15.0 mg/L) were all significantly higher than in samples from healthy individuals (32.3 ± 8.8 mg/L). TTR levels in vitreous and corresponding serum samples It was noteworthy that TTR could be detected in all vitreous samples and the corresponding serum samples. The TTR concentration of high myopia patients with macular detachment and macular hole was significantly higher in both vitreous and corresponding serum samples. However, the TTR concentration in vitreous samples from patients with long axis lengths was lower than in the corresponding serum samples (p N 0.05) (Table 4). Visual acuity was well correlated with vitreous TTR. We found that patients with better visual acuity post-operatively had higher TTR concentrations in vitreous and lower concentration in serum (Table 5). Thus, TTR may be a promising candidate serum biomarker for the evaluation of different ocular pathologies and visual acuity. Discussion We found that TTR levels were higher in serum samples from patients with high myopia, especial in patients with ocular pathologies. The TTR concentration of serum samples in the high myopia patients with macula detachment, macular hole, choroidal neovascularization, macular epimacular membrane, and atrophy (235.5 ± 23.0 mg/L, 131.9 ± 15.0 mg/L, 94.0 ± 6.0 mg/L, 84.1 ± 2.0 mg/L, and 70.6 ± 3.5 mg/L respectively) were dramatically higher than samples from healthy patients (32.3 ± 8.8 mg/L). These results underscore the high correlation between elevated serum TTR and high myopia patients with ocular pathology. Studies on the pathogenesis of high myopia indicate that genetic influences played a significant role in its
Table 1 The significantly differential proteins in hereditary high myopia patients' serums. Differential proteins
Protein occurrence frequency in 4 healthy serums' flow-through fractions
Protein occurrence frequency Description in 16 abnormal serums' flow-through fractions
Hemopexin Apo B-100 Transthyretin (TTR)
1 1 0
16 12 16
Haptoglobin (HP)/Haptoglobin-related 0 protein (HPr)
16
The relationship with high myopia is unclear. Associated with age-related maculopathy Associated with vitreous amyloidosis, nerve amyloid, ocular amyloid angiopathy, and other ocular related disease. Associated with self immunization; higher serum haptoglobin levels in patients with Behcet's disease (an ocular related disease) compared to control subjects were obtained.
684
J. Shao et al. / Clinical Biochemistry 44 (2011) 681–685
Fig 1. The levels of TTR were detected in serum of normal individuals and high myopia patients by western blot. A: 1, 2, 9, 10 represented TTR levels of macular hole patients in serum; 3, 4, 11, 12 represented TTR levels of central fovea of macula detachment patients in serum; 5, 6 represented TTR levels of atrophy patients in serum; 7, 8 represented TTR levels of normal individuals in serum. 13, 14 represented TTR levels of choroidal neovascularization patients, 15 represented TTR levels of macular epimacular membrane patients in serum, 16 represented TTR levels of patients with no significant ocular pathology in serum. B: 1, 2 represented TTR levels of AL was 30 mm or more ; 3, 4 represented TTR levels of AL ranging from 28 mm to 29.99 mm; 5, 6 represented TTR levels of AL ranging from 26.0 mm to 27.99 mm ; 7, 8,13,14,15,16 represented TTR levels of normal individuals in serum. 9–12 represented TTR levels of high myopia patients in serum. In conclusion, TTR level in serum increased accompany by growth of AL; each pathologic appearance of TTR level in serum was significantly different.
development [12]; however, genetic analysis provided an incomplete picture. Proteomic analysis can provide a more detailed picture of the relationship between gene sequences and cellular physiology [13], and so can complement genetic analysis for evaluating disease development, prognosis, and response to treatment [14]. Our LQTMASS results demonstrate the value of identifying and detecting protein biomarkers for disease, in this case for high myopia, that can be employed for diagnosis and monitoring. Consistent with the findings of van Aken et al. [15], our results also demonstrated that visual acuity was positively correlated with vitreous TTR concentrations. The average vitreous TTR concentration of patients with visual acuity N 0.1 (140.5 ± 4.5 mg/L) was significantly higher than in patients with visual acuity b 0.1 (89.5 ± 5.5 mg/L). A higher metabolic rate in cells of the retinal pigment epithelium (RPE) in high myopia patients with better visual acuity may account for this higher TTR levels. The RPE of the eye and choroid plexus of the brain are developmental and functional homologues, sharing many of the characteristics of transporting epithelia; cells are joined by tight apical membrane junctions and they regulate the transport of fluids and serum proteins into their respective humors. The expression of TTR mRNA in RPE cells is higher than all other tissues except for the choroid plexus [16]. The TTR protein is secreted predominantly in an apical direction by RPE cells in vitro [17]. The source of all-transretinol for the RPE is the liver, which secretes retinol bound to the TTR–RBP complex. In RPE cells, free retinol uptake appears to be mediated by STRA6, a retinol-RBP cell membrane receptor that is highly expressed on the basolateral side of these cells [18]. The physiological role of TTR in the eye is not completely known, but it may participate in retinol cycling. The fact that retinol metabolism
Table 2 TTR concentrations of high myopia patients’ serum samples. Clinical parameter Visual acuity
Axial length
Pathological appearance
N 0.5 0.1–0.5 b 0.1 N 30 28–30 26–27.99 macular detachment macular hole choroidal neovascularization macular epimacular membrane Atrophy no significant pathological changes
was altered in TTR-null mice suggests that TTR plays a major role in retinol metabolism in the interphotoreceptor matrix (IPM) [19]. Almost all eye diseases are coupled with destruction of bloodretina barriers. It was reported that high myopia patients with ocular pathologies showed accompanying changes in blood-retinal barrier permeability [20]. In the present report, the mean TTR concentrations of serum samples in patients with macular hole and macular detachment (109.3 ± 24.5 mg/L, 165.5 ± 25.5 mg/L) were similar to the vitreous concentrations (99.2 ± 23.5 mg/L, 121.9 ± 25.5 mg/L), but still much higher than the average TTR concentration from vitreous samples from healthy subjects (44.5 ± 7.5 mg/L). Macular hole and macular detachment are thought to be caused by traction of the vitreous, long axial length, posterior staphyloma, and RPE atrophy. In response to these kinds of damage, TTR in the vitreous might flow into the serum from cells and tissues of the eye through damaged blood-retina barriers. To maintain vitreous TTR, high rates of synthesis might be required, thus explaining the high vitreous TTR concentrations in these conditions. Furthermore, the high serum TTR concentrations may be associated with destruction of blood–retina barriers. Retinal and choroid atrophy are usually caused by continuous extension of the sclera posterior pole, and the RPE denaturizing for retinal and choroidal vascular distortion [21]. Retinal scars and choroid atrophy are often detected in the development of late high myopia [22]. Irreversible RPE damage should lower serum TTR concentrations. However, the serum TTR concentration of patients with macular epimacular membrane was higher than samples from health individuals because of increased TTR secretion caused by proliferation of RPE cells and Müller glial [23]. Additionally, the increased level of TTR in the retinal blood circle could stimulate the proliferation of RPE. The TTR concentration of serum samples in high myopia patients with choroidal neovascularization (94.0 ± 6.0 mg/L) was upregulated. It was reported that estrogens and androgens up-regulate TTR gene expression in liver and choroid plexus in mammals [24]. Estrogen receptors were also found in bovine retina and especially on vessels in the choroid [25,26]. Recently, several studies demonstrated the expression of estrogen receptors in human retina and retinal pigment epithelium [27,28]. Estrogen receptors were reported to be expressed in the CNV of highly myopic eyes [29]. Transthyretin might stimulate estrogen receptors to increase TTR synthesis. The average TTR
Number of patients
TTR concentration
p Value
28 50 38 31 37 48 17 14 20
112.4 ± 36.5 mg/L 97.6 ± 44.5 mg/L 108.7 ± 34.0 mg/L 182.0 ± 28.0 mg/L 98.0 ± 15.0 mg/L 60.5 ± 9.5 mg/L 235.5 ± 23.0 mg/L 131.9 ± 15.0 mg/L 94.0 ± 6.0 mg/L
N 0.05
11
84.1 ± 2.0 mg/L
Age
28 26
70.6 ± 3.5 mg/L 55.0 ± 3.5 mg/L
Sex
b 0.01
b 0.01
Table 3 TTR concentrations in controls' serum samples. Age/sex b 40 40 ~ 50 N 50 female male
Number of controls
TTR concentration
p Value
20 56 10 44 42
27.8 ± 3.2 mg/L 35.9 ± 9.3 mg/L 21.1 ± 5.8 mg/mL 33.9 ± 5.3 mg/L 31.6 ± 4.7 mg/L
N 0.05
N 0.05
J. Shao et al. / Clinical Biochemistry 44 (2011) 681–685
References
Table 4 TTR concentration in serum and vitreous samples. Clinical parameter
Number of Concentration of TTR in patients different samples
Pathological 40 appearance Macular detachment 18 Macular hole 22 None 8 Axial length N 30 28–30 26–27.99 b 26
18 12 10 8
vitreous
685
p Value
serum
121.9 ± 25.5 mg/L 165.5 ± 25.5 mg/L N 0.05 99.2 ± 23.5 mg/L 109.3 ± 24.5 mg/L N 0.05 44.5 ± 7.5 mg/L 33.0 ± 9.0 mg/L N 0.05
129.8 ± 12.0 mg/L 188.5 ± 15.5 mg/L 96.0 ± 7.0 mg/L 115.0 ± 6.0 mg/L 88.5 ± 12.0 mg/L 60.5 ± 3.5 mg/L 45.6 ± 5.6 mg/L 33.5 ± 4.3 mg/L
N 0.05 N 0.05 N 0.05 N 0.05
Table 5 The association between visual acuity postoperatively and TTR concentration in serum and vitreous samples. Visual acuity postoperatively
Number of patients
Vitreous
Serum
N 0.5 0.1–0.5 b 0.1 p
0 15 25
0 140.5 ± 4.5 mg/L 89.5 ± 5.5 mg/L p b 0.05
0 96.4 ± 12.7 mg/L 157.8 ± 11.5 mg/L p b 0.05
concentration of serum samples in patients with no significant ocular pathologies (55.0 ± 3.5 mg/L) was lower than in high myopia cases with ocular pathologies, suggesting that TTR might be a promising candidate serum biomarker for evaluation the risk for ocular pathologies in high myopia patients. TTR has been associated with a number of pathological conditions, including Alzheimer's disease, Parkinson's disease, schizophrenia, and depression. Other reports suggest that TTR was associated several types of cancer, diabetes, rheumatoid arthritis, glomerular disease, and hepatitis. Moreover, TTR is thought to be directly and causally involved in the establishment of all stages of the stress response to nutritional impairment and in acute stressful conditions, such as tissue injury. Thus, TTR is also as an indicator of malnutrition. In our study, we eliminated these factors by the strict selection of subjects. TTR is an age-associated protein [30]. We discriminated the effect of age and of clinical symptoms of TTR levels by analyzing TTR concentration of normal group. In normal group, our experiment showed that TTR levels of the serum of subjects younger than 40 years old, 40–50 years old, and over 50 years old were 27.8 ±3.2 mg/L, 35.9± 9.3 mg/L and 21.1 ± 5.8 mg/mL, respectively, there were no differences in physical conditions within the different age group. So it indicated that age is not a significant factor influencing the difference in serum TTR concentrations between ages and clinical symptoms. We showed that TTR concentrations in the vitreous samples (129.8 ± 12.0 mg/L) of patients with long AL (N 30 mm) were lower than in the corresponding serum samples (188.5 ± 15.5 mg/L). High myopia patients with long axial length had poor visual function [31], indicating that low TTR may be used as a biochemical marker for assessing visual disturbances. It is noteworthy that TTR may be one of the candidate serum biomarkers of high myopia. However, confirmation still requires large-scale studies. Further research is also needed to examine the role of TTR in the pathogenesis of ocular diseases and to develop new methods for the prevention and control of high myopia. Acknowledgments This work was supported by the program of the Key Laboratory of fundus oculi diseases, Shanghai, China (No. YDB-0901).
[1] Grossniklaus HE, Green WR. Pathologic findings in pathologic myopia. Retina 1992;12:127–33. [2] Lam CS, Goldschmidt E, Edwards MH. Prevalence of myopia in local and international schools in Hong Kong. Optom Vis Sci 2004;81:317–22. [3] Avila MP, Weiter JJ, Jalkh AE, Trempe CL, Pruett RC. Natural history of choroidal neovascularization in degenerative myopia. Ophthalmology 1984;91:1573–81. [4] Xu L, Wang Y, Li Y, Wang Y, Cui T, Li J, Jonas JB. Causes of blindness and visual impairment in urban and rural areas in Beijing. The Beijing Eye Study. Ophthalmology 2006;113:1134–41. [5] Wong TY, Foster PJ, Hee J, Ng TP, Tielsch JM, Chew SJ, Johnson GJ, Seah SK. Prevalence and risk factors for refractive errors in adult Chinese in Singapore. Invest Ophthalmol Vis Sci 2000;41:2486–94. [6] Herbert J, Wilcox JN, Pham KC, Fremeau RT, Zaviani M, Dwork A, Soprano D, Makover A, Goodman DS, Zimmerman EZ, Roberts JL, Schon EA. Transthyretin: a choroid plexus specific transport protein in human brain. Neurology 1986;36:900–11. [7] Jacobsson B, Collins VP, Grimelius L, Pettersson T, Sandstedt B. Carlstrom A Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic waslets. J chem Cytochem 1989;37:31–7. [8] Monaco HL, Rizzi M, Coda A. Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 1995;268:1039–41. [9] Delcourt C, Dupuy AM, Carriere I, Lacroux A, Cristol JP. Albumin and transthyretin as risk factors for cataract: the POLA study. Arch Ophthalmol 2005;123:225–32. [10] Mullins RF, Russell SR, Anderson DH, Hageman GS. Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. FASEB J 2000;14:835–46. [11] Duan X, Lu Q, Xue P, Zhang H, Dong Z, Yang F, Wang N. Proteomic analysis of aqueous humor from patients with myopia. Mol Vis 2008;14:370–3. [12] Jacobia FK, Zrennera E, Broghammerb M, Puschb CM. A genetic perspective on myopia cell. Mol Life Sci 2005;62:800–8. [13] Young TL, Metlapally R, Shay A. Complex trait genetics of refractive error. Arch Ophthalmol 2007;125:38–48. [14] Dove A. Proteomics: translating genomics into products? Nat Biotechnol 1999;17: 233–6. [15] Van Aken E, De Letter EA, Veckeneer M, Derycke L, van Enschot T, Geers I, Delanghe S, Delanghe JR. Transthyretin levels in the vitreous correlate with change in visual acuity after vitrectomy. Br J Ophthalmol 2009;93:1539–45. [16] Pfeffer BA, Becerra SP, Borst DE, et al. Expression of transthyretin and retinol binding protein mRNAs and secretion of transthyretin by cultured monkey retinal pigment epithelium. Mol Vis 2004;10:23–30. [17] Ong DE, Davis JT, O'Day WT, et al. Synthesis and secretion of retinol-binding protein and transthyretin by cultured retinal pigment epithelium. Biochemistry 1994;33:1835–42. [18] Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, Wiita P, Bok D, Sun H. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 2007;315:820–5. [19] Palha JA, Nissanov J, Fernandes R, Sousa JC, Bertrand L, Dratman MB. Thyroid hormone distribution in the mouse brain: the role of transthyretin. Neuroscience 2002;113:837–47. [20] Kitaya N, Ishiko S, Abiko T, Mori F, Kagokawa H, Kojima M, Saito K, Yoshida A. Changes in blood–retinal barrier permeability in form deprivation myopia in tree shrews. Vis Res 2000;40:2369–77. [21] Miller DG, Singerman LJ. Natural history of choroidal neovascularization in high myopia. Curr Opin Ophthalmol 2001;12:222–4. [22] Ohno-Matsui K, Yoshida T, Futagami S, Yasuzumi K, Shimada N, Kojima A, Tokoro T, Mochizuki M. Patchy atrophy and lacquer cracks predispose to the development of high myopia of choroidal neovascularisation in pathological myopia. Br J Ophthalmol 2003;87:570–3. [23] Garweg JG, Bergstein D, Windisch B, Koerner F, Halberstadt M. Recovery of visual field and acuity after removal of epiretinal and inner limiting membranes. Br J Ophthalmol 2008;92(2):220–4. [24] Tang YP, Haslam SZ, Conrad SE, Sisk CL. Estrogen increases brain expression of the mRNA encoding transthyretin, an amyloid beta scavenger protein. J Alzheimers Dis 2004;6:413–20. [25] Goncalves I, Alves CH, Quintela T, Baltazar G, Socorro S, Saraiva MJ, Abreu R, Santos CR. Transthyretin was up-regulated by sex hormones in mice liver. Mol Cell Biochem 2008;317:137–42. [26] Kobayashi K, Kobayashi H, Ueda M, Honda Y. Estrogen receptor expression in bovine and rat retina. Invest Ophthalmol Vis Sci 1998;39:2105–10. [27] Ogueta SB, Schwartz SD, Yamashita CK, Farber DB. Estrogen receptor in the human eye: influence of gender and age on gene expression. Invest Ophthalmol Vis Sci 1999;40:1906–11. [28] Wickham LA, Gao J, Toda I, Rocha EM, Ono M, Sullivan DA. Identification of androgen, estrogen and progesterone receptor mRNAs in the eye. Acta Ophthalmol Scand 2000;78:146–53. [29] Kobayashi K, Mandai M, Suzuma I, Kobayashi H, Okinami S. Expression of estrogen receptor in the choroidal neovascular membrane in highly myopic eyes. Retina 2002;22:418–22. [30] Ando Yukio, Nakamura Masaaki, Araki Shukuro. Transthyretin-related familial amyloidotic polyneuropathy. Arch Neurol 2005;62:1057–62. [31] Nakanishi H, Kuriyama S, Saito I, Okada M, Kita M, Kurimoto Y, Kimura H, Takagi H, Yoshimura N. Prognostic factor analysis in pars plana vitrectomy for retinal detachment attributable to macular hole in high myopia: a multicenter study. Am J Ophthalmol 2008;146(2):198–204.