|Year : 1999 | Volume
| Issue : 1 | Page : 31-34
Transthyretin (prealbumin) in eye structures and variation of vitreous-transthyretin in diseases
S Ramakrishnan, KN Sulochana, Sunil Parikh, R Punitham
Biochemistry Research Department, Vision Research Foundation, Chennai, India
Vision Research Foundation, 18 College Road, Chennai - 600 006
Source of Support: None, Conflict of Interest: None
Purpose: To evaluate the presence of transthyretin (TTR, prealbumin) a protein which binds retinol to retinol-binding protein in various ocular tissues and to study its quantitative changes in the vitreous humor in various diseases
Method: Estimation of TTR was done by electrophoresis of 10 mg protein in each sample of tears, aqueous humor, vitreous, retina, and lens by an Imaging Densitometer using prealbumin as the standard.
Results: TTR was present in all the eye structures except the lens and tear. The retina and the vitreous had relatively higher amounts of TTR compared with aqueous. The identity of TTR was confirmed by immuno-electrophoresis using anti-human TTR. Two bands in SDS electrophoresis revealed that this protein is a heterodimer. There was a significant decrease in vitreous TTR in diabetes with hypertension and increase in one case each of diabetes with hypertension associated with leukaemia or carcinoma with hepato-splenomegaly.
Conclusion: Vitreous TTR is probably from retina and retinal pigment epithelium. The level of vitreous TTR is likely to have diagnostic significance in some retinal diseases.
Keywords: Transthyretin, eye structures, biochemical function, vitreous transthyretin in diseases
|How to cite this article:|
Ramakrishnan S, Sulochana K N, Parikh S, Punitham R. Transthyretin (prealbumin) in eye structures and variation of vitreous-transthyretin in diseases. Indian J Ophthalmol 1999;47:31-4
Transthyretin (Prealbumin) (TTR) is a protein which binds retinol to retinol-binding protein. Normal blood level is 25 to 30 mg/dL., Tears are reported to have Tear Specific Prealbumin (TSP).[3-6] Biosynthesis of TTR occurs in the pigment epithelium and liver. In 2 cases of amyloid, TTR variant was found in the vitreous. Hence it was of interest to analyse vitreous and other structures of the eye such as the aqueous, retina and tear to detect the presence, and if present, to measure the level of TTR in ocular structures in health and disease.
| Materials and Methods|
Tear fluids were collected from individuals with no complaint of ocular illness, from the study centre (Sankara Nethralaya, Chennai). The volunteers in age group 20-30 years were from the clinical and biochemistry research laboratories. Weakly stimulated reflex tear samples (10-50 ml) were taken from the outer lower part of the conjunctival sac using 25 ml glass capillaries, and were stored separately at -20° C until use.
| Aqueous|| |
Aqueous humor samples were collected from community ophthalmology patients of age 50-60 years who underwent extra capsular cataract extraction (ECCE) with or without intraocular lens (IOL) implantation at Sankara Nethralaya. A tuberculin syringe with a 25 G needle was used to draw 0.1ml of aqueous samples just before surgery, and was transported to the Biochemistry Research laboratory.
| Lens, vitreous retina|| |
Vitreous samples used in this analysis were either from donor eyeballs or from patients of 50-60 years who underwent vitrectomy. Lens and retina samples were from human donor eyes.
| Lens and retina homogenate|| |
One hundred milligrams of lens matter, was homogenized in 1 ml of phosphate buffer (pH 7.4) and centrifuged for 30 minutes at 10,000 rpm. The clear supernantant was used for protein estimation. For immuno-electrophoresis 200 mg of lens protein was used.
Retina homogenate was prepared as for the lens. For immuno-electrophoresis and poly acrylamide gel electrophoresis (PAGE). 20 mg and 10 mg protein were used respectively.
Immuno-electrophoresis was carried out by the method of Clarke and Freeman using 1% agarose on microscopic glass plates. Electrophoresis was performed using 20, 20, 200, 20, and 20 mg of tear, aqueous, lens, vitreous, and retinal protein respectively. Vitreous with a higher concentration of protein, serum and standard pre albumin (Sigma) were also analysed. At the end of electrophoresis, a well 30 mm length x 3 mm breadth was set, and 28 mg of anti-human TTR (Sigma) was loaded. The slides were kept in a moist chamber for 24 - 36 hours. At the end of the incubation period, they were removed, dried at 60° C for 1 hr. and stained for protein using Commassive brilliant blue R-250 and destained by acetic acid, methanol, and water mixture.
Samples of tear, aqueous humor, vitreous humor, and retinal-homogenate, each containing 10 mg of proteins and 5 mg prealbumin (Sigma) were loaded separately in 9% polyacrylamide gel. The TTR concentration was determined using a densitometer, after staining the gel with silver stain.
The protein was treated with sodium dodecyl sulphate (SDS) to find out whether it is a monomeric or polymeric protein and SDS PAGE was done using 12% gel with Sigma SDS molecular weight markers (Bovine serum albumin 66,000; Egg albumin 45,000; Glyceraldehyde 3; phosphate dehydrogenase 36,000; Carbonic anhydrase 29,000; Trypsinogen 24,000; Trypsin-inhibitor 20,100; and Lactalbumin 14,200).
Vitreous TTR studies were done on 15 samples from eye donors who served as controls and 28 patients who underwent vitreous surgery. Details of number of patients in the second group, and their eye diseases are given in the Table. Ten patients did not have any specific complaints. Vitreous TTR was analysed in all the samples after equal intervals between collection from the donor eye and surgery on the patients.
To eliminate contamination with blood, serum and vitreous samples were simultaneously subjected to PAGE and the albumin:TTR ratio was determined using Bio Rad's GS-670 imaging densitometer. The ratio between the TTR in serum and vitreous humor and the concentration of TTR in different samples of vitreous humor was also determined after PAGE using the same densitometric equipment.
Protein in samples was estimated by the methods of Lowry et al using bovine serum albumin as standard.
| Results|| |
All the examined structures of the human eye except lens and tears had the same TTR.
In immunological analysis, prealbumin (Sigma), serum, aqueous humor, vitreous humor and retinal samples gave precipitin arcs with anti-human TTR [Figure - 1], confirming the identity of TTR in these samples. There were no such arcs in the lens samples and tears [Figure - 1]. The PAGE of tears showed, in addition to tear specific prealbumin (TSP), another one with a slightly different RF of human TTR. This protein appeared to be different from TTR of serum or other eye structures. Absence of precipitin arcs in tear proteins with anti-human TTR confirms this important finding.
SDS treatment of native TTR followed by electrophoresis revealed 2 separate bands, one at 17.8 KDa and the other at 34 KDa [Figure - 2]. Similar SDS treatment of vitreous samples also revealed these two proteins [Figure - 2].
The ratio of albumin to TTR in serum was nearly 100 : 1 while that of vitreous 5 : 1. For this study, 5 samples of serum and 5 vitreous from 5 donor eyeballs were used.
The levels of TTR were almost the same in donor eye samples, and patients with no specific complaints. The values of TTR/10 mg protein of aqueous, vitreous and retina ranged from 0.53 to 0.64, 1.28 to 1.31, and 0.85 to 0.89 mg respectively [Figure - 3].
There was a decrease of vitreous TTR in some diseases, (Table) especially diabetes with hypertension. The decrease was statistically significant. Interestingly, if diabetes with hypertension was associated with leukaemia, or carcinoma with hepato-splenomegaly, there was a phenomenal increase of vitreous TTR.
| Discussion|| |
There are no definite observations in the literature that TTR is present in different structures of the eye. The present study records the presence of TTR in all the structures of the eye except lens and tears [Figure - 1]. This observation is interesting as it raises questions on the role of TTR in retina, vitreous and the aqueous.
Retina and vitreous have relatively higher amounts of TTR [Figure - 3]. The presence of TTR in the retina and the vitreous suggests new biochemical and physiological functions of the protein.
Retinol from the liver is bound to retinolbinding-protein (RBP) and to TTR; this complex is transported in blood. From the capillaries of the retina, retinol alone moves into the rods while the two associated proteins continue their circulation in the blood to get eliminated through the kidneys. The question arises then as to how TTR is found in the retinyl extract. It is perhaps the protein synthesized in pigment epithelium. Mizuno et al have proposed that ocular TTR may play a role in the intraocular translocation of retinol to the retina for use in the visual cycle. This was supported by their detection of RBP and RBP mRNA in rat retinal pigment epithelium (RPE).
In addition, there may be another role for the TTR of RPE. It is well known that when light falls on rhodopsin, all trans-retinol formed moves into the pigment epithelium. This protects the disc membrane from the alcoholic effects on retinol and its denaturation.
In RPE, it is reversibly converted to retinyl ester, as an ester, unlike alcohol, it may not exert a necrotic effect on the biomembrane. To impart additional safety to the rod membrane and to minimise loss of vitamin A, the RPE appears to synthesise TTR which might bind the vitamin and its derivative. In dark, when rhodopsin synthesis is metabolically warranted, TTR of RPE may release vitamin A from its fold. This proposed sequence of events also may justify the synthesis of TTR by RPE, and the presence of significant amounts of TTR in the retina.
In addition to retina, the vitreous also has significant amounts of TTR. The question arises as to whether (i) TTR is the product of synthesis from hyalocytes, (ii) has been received from the blood, or (iii) has reached vitreous from retina and the usual wear and tear of RPE.
If the role of TTR is to transport and perhaps bind and preserve vitamin A, it has apparently no role in the vitreous. Hence, it is unlikely that it might be synthesized by the hyalocytes, as the living system does not indulge in any wasteful anabolism in the interests of economy. The present work has shown that vitreous TTR is not from the blood as the albumin:TTR ratio in serum is about 100:1 while that in the vitreous is 5:1. This clearly shows that there is no contamination of vitreous with blood. It is quite likely that the TTR of vitreous is from the retina and RPE for the following reasons. Gorevic et al report that the bulk of the protein constitutents of normal vitreous is produced by the retina with prealbumin as a significant normal constitutent of soluble proteins. Reddy proposes that the vitreous can act as a metabolic repository for retinal protein metabolism. Such metabolism has been cited as the reason that vitreous levels of glutamic acid are similar to plasma, a phenomenon attributed to the release of the substance by retinal metabolism. As a corollary, it is possible that in some diseases of the retina, the TTR level of vitreous may have diagnostic significance.
Soltau et al report that in 2 cases of amyloid with vitreous opacity, they were able to confirm the clinical diagnosis of amyloid by finding a variant of TTR. In the present work, we find that the levels of vitreous TTR were decreased in diabetes with hypertension but phenomenally increased if the same was accompanied by leukaemia or carcinoma. It appears that in cancer, the levels of vitreous TTR are increased.
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]