|Year : 1988 | Volume
| Issue : 1 | Page : 7-9
Ascorbic acid estimation in aqueous humour of normal cataractous and aphakic eyes
Safina Church Road, Near State Garage, Jahangirabad Bhopal (MP), India
M A Khurrum
Safina Church Road, Near State Garage, Jahangirabad Bhopal (MP)
Source of Support: None, Conflict of Interest: None
Ascorbic acid has received a great deal of attention in lens metabolism. A good number of workers have estimated aqueous humour and serum ascorbic acid values in animals and human beings. But the question whether the concentration of ascorbic acid in aqueous humour varies in normal, cataractous and aphakic patients is still disputed.
|How to cite this article:|
Khurrum M A. Ascorbic acid estimation in aqueous humour of normal cataractous and aphakic eyes. Indian J Ophthalmol 1988;36:7-9
| Introduction|| |
Carbohydrate is the main source from which the lens derives its energy. But the consumption of oxygen by the lens is too small to account for the metabolism of carbohydrate. There has always been speculation as to whether some hydrogen acceptor other than oxygen might be used. The high concentration of glutathione and ascorbic acid in the lens and aqueous humour have encouraged this belief since both of these substances are capable of being irreversibly reduced and oxidized under physiological conditions
| Materials and Methods|| |
Estimation of ascorbic acid in Aqueous humour was done in thirty cases; constituting 10 normal, 10 mature cataractous and 10 aphakic subjects, who were admitted in the in patient department of ophthalmology, Hamidia Hospital, Bhopal.
Criteria for selection of normal cases
This group of normal cases was selected from two types of patients admitted in the eye wards:
1. Patients with chronic simple glaucoma without any lenticular opacity. Aqueous humour was taken at the time of surgery.
2. Patients with a mature cataract in one eye and no lenticular opacity in the other eye. Aqueous humour was aspirated from the normal eye.
Criteria for selecting mature cataractous and aphakic cases
Ascorbic acid estimation in aqueous humour was also done in 10 mature cataractous and 10 aphakic cases.
Certain special investigations were done to confirm the diagnosis of a fully mature colorab viz oblique illumination, fundoscopy and slit lamp microscopy.
The patients were prepared as for an intraocular operation A tuberclin syringe with number 26 hypodermic needle was used for the aquerous top. The eyeball was fixed by fixation forceps at 6 O'clock position so that its tip just appeared in the anterior chamber. The fluid was withdrawn very slowly till the anterior chamber completely collapsed. The fluid was immediately transferred to a dry test tube. The anterior chamber was reformed by injecting air or saline.
The operation for glaucoma or cataract was then carried out as usual.
Method of determination of ascorbic acid in aqueous humour
The 2-4 Dinitrophenylhydrozine method of Roe and Kuether (1943) for estimation of ascorbic acid in plasma was used
Principle: The 2-4 Dinitrophenylhydrazine method estimates ascrobic acid after oxidation. The plasma is deproteinized with trichloroacetic acid and shaken with activated charcoal in order to oxidize the ascorbic acid to dehydroascorbic acid By treatment with Dinitrophenylhydrazine in the presence of thiourea an intensely red coloured bishydrazone is formed which is estimated colorimetrically after treatment with concentrated sulphuric acid.
Method : One ml of plasma (i e. aqueous diluted to one ml) is treated with 4 ml of 7% trichloroacetic acid and thoroughly shaken. 0.15 gm of activated charcoal is added after 5 minutes and mixed well again After 10 minutes the proteins and the charcoal are centrifuged down land the supernatant passed through a filter paper.
Test: 2 ml of filtrate (= 0.4 ml of diluted aqueous humour) is taken in a tube marked at 5 ml, and treated with 0.1 ml thiourea and 0.5 ml of Dinitrophenylhydrazine. The tube is stoppered with a rubber bung and kept at 37 oC for 3 hours. The tube is then placed in ice water and 1 ml of sulphuric acid is added dropwise over a period of one minute while the solution is stirred with a glass rod.
The solution is kept for 30 minutes at the end of which an intensely red when develop. Water is added to the 5 ml mark and mixed.
Standards: Three standards are prepared corresponding to 0.5 mg, 1.0 mg and 2.0 mg ascorbic acid per 100 ml as follows:
10 ml of working standards are treated with 0.3 gm activated charcoal in the same way as with plasma (ie. aqueous) and trichloroacetic acid and Shaken This is filtered after 5 minutes. 0.5 ml, 1.0 ml and 2.0 ml of this working standard (= 0.002, 0.004 and 0.008 mg ascorbic acid) are then diluted to 2 ml with water and then treated with thiourea, Dinitrophenylhydrazine and sulphuric acid in the same way as the test.
Blanks : (1) For the test- The blank is prepared in the same way as the test except that the Dinitrophenylhydrazine is added after sulphuric acid.
(2) For the standards - 2 ml of water are used instead of 2 ml of ascorbic acid solution, the other reagents are the same.
The colour of the test and the appropriate standards are red in a. photoelectric colorimeter. The reading of the blanks are subtracted from those of the respective test and standard A green 624 light filter should be used. The standard whose colour is most nearly equal to that of the test is used.
The results are multiplied by the dilution factor.
| Observations|| |
The present study consists of estimation of ascorbic acid level in the aqueous humour of 10 normal healthy individuals, 10 mature cataractous and 10 aphakic patients, admitted in the patient department of ophthalmology, Hamidia Hospital Bhopal.
| Discussion|| |
The consumption of carbohydrate by the lens is at the rate of about 0.14 mgm glucose/hour/rabbit lens. It is agreed that only a small fraction of glucose utilised by the lens is broken down by aerobic processes, estimated as being between 14-30% (Kinoshita and Wacht 1958). There has always been speculation, however, as to whether some hydrogen acceptor other than oxygen might be used. The high concentration of ascorbic acid in a queous humour and lens and of glutathione in lens have supported this belief: since both of these substances are capable of being reversibly reduced and oxidized under physiological conditions, but there is no evidence of thier participation in the oxidative mechanism of the lens.
An attempt has been made to evaluate the role of ascorbic acid in the metabolism of the lens and to study the various factors responsible for relatively higher concentration of ascorbic acid in the aqueous humour than in the serum.
The results obtained were compared with that of other observers and all the values recorded have been subject to statistical analysis,
The following conclusions are drawn:
1. The concentration of ascorbic acid in the aqueous humour in Indian patients with mature cataract and aphakia is found to be more or less same as compared with that in normal cases, The mean values of ascorbic acid in aqueous humour detected are
Normal 5.7 mgm%
Mature cataract 5.5 mgm%
Aphakia 4.43 mgm%
2. The concentration of ascorbic acid in aqueous humour in normal, mature cataract and aphakia uses is lower as compared with white people. The concentration of ascorbic acid in aqueous humour of white people is about 18-20 mgm% (Purcell et al, 1954). In our study the average value is about 5 mgm%.
3. The lens has got no definite role in the synthesis of ascorbic acid in the aqueous humour and it is not solely responsible for the maintenance of relatively higher concentration of the substance in the aqueous humour.
4. The high concentration of ascorbic acid in the aqueous humour is probably due to secretion/diffusion mechanism from the ciliary epithelium.
5. The exact role of ascorbic acid in the metabolism of the lens still remains obscure.
| References|| |
Adler F. R, Physiology of the eye, 5th edition, page no. 307, 340, 348, Henry Kimpton, London, 1970
Chatterjee O.M and Gosh B.P., Arch Ophthalmol, page no. 46, 1956
Day P.L American J. Ophthalmol, 14; 1005, 1931.
Ely LO. & Robbie, American J. OphthalmoL 33; 269, 1950.
Friedenwald J.S, Arch Ophthalmol 29; 535, 1943.
Kinsey V.H, American J. Ophthalmol 33; 257, 1950.
Langham ME & Heald K, Biochemical J. 63; 52, 1956.
Uyama V. and Ugino, Med. J. Osaka Univ. 7; 327, 1956.
[Table - 1], [Table - 2], [Table - 3], [Table - 4]