Year : 2000 | Volume
: 48 | Issue : 1 | Page : 37--43
Possible role of polyamines in gyrate atrophy.
KN Sulochana, S Ramakrishnan, L Mahesh, R Punitham
Biochemistry Research Department, Vision Research Foundation, 18 College Road, Chennai-600 006, India
K N Sulochana
Biochemistry Research Department, Vision Research Foundation, 18 College Road, Chennai-600 006
PURPOSE: Gyrate atrophy (GA) is marked by hyperornithinemia and lowered ornithine amino transferase (OAT). However there are patients of GA without hyperornithinemia and those with hyperornithinemia without GA. Some cases of GA have been reported to have low lysine. The purpose of the study was to determine if polyamines, the metabolites of ornithine, and lysine have any diagnostic role in GA. METHODS: Ornithine in plasma was estimated by two-dimensional paper chromatography, with elution of the coloured spot, and the absorbance measured using a spectrophotometer at 560 nm. OAT assay in lymphocytes was done spectrophotometrically using ornithine as substrate. Blood and urinary polyamines were extracted with n-butanol, benzoylated and analysed with HPLC; putrescine, spermine, spermidine, and cadaverine were assayed individually at 254 nm with the UV detector using ODS, G18 column with 63% methanol as solvent. RESULTS: Of the 7 patients investigated, 6 had features typical of GA. One was diagnosed to have atypical retinitis pigmentosa (case 3). The first five cases had elevated ornithine and diminished OAT, but cases 6 and 7 had near-normal ornithine and case 7 had near-normal OAT. However, all 7 patients had increased levels of total polyamines in urine compared to normals. Five had increased putrescine and three had increased spermine. All the 7 had decreased cadaverine in urine. Thus, though there were inconsistencies with ornithine and OAT, all the 7 patients had elevated polyamines from ornithine and decreased cadaverine. CONCLUSION: In addition to estimating ornithine and OAT in GA, it is suggested that urinary polyamines may be analysed as the latter appears to correlate better with the clinical condition and help in the diagnosis to a greater extent. Moreover, while ornithine is an innocuous amino acid, polyamines are known to damage DNA and proteins.
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Sulochana K N, Ramakrishnan S, Mahesh L, Punitham R. Possible role of polyamines in gyrate atrophy. Indian J Ophthalmol 2000;48:37-43
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Sulochana K N, Ramakrishnan S, Mahesh L, Punitham R. Possible role of polyamines in gyrate atrophy. Indian J Ophthalmol [serial online] 2000 [cited 2021 Jun 18 ];48:37-43
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Gyrate atrophy (GA) of the choroid and retina is chorioretinal degeneration with an autosomal recessive mode of inheritance. The term "gyrate" (Latin: turned round) was originally used to describe this disease because the margin of the retinal atrophy in the early stages of the disease curves as circular segments. Patients report night blindness and loss of peripheral vision between the ages of 10 and 20. Ocular findings include myopia, constricted visual fields, elevated dark adaptation thresholds, very little or no electroretinographic responses and chorioretinal atrophy distributed circumferentially around the peripheral fundus and often near the disc. In more advanced stages, the areas of peripheral chorioretinal atrophy coalesce and extend posteriorly, and patients develop progressive constriction of the visual field, cataracts, and eventual blindness between ages 40 and 50. It has been reported that the plasma ornithine concentration in affected patients is 10 to 20-fold above normal.[1-3] The finding of this association between hyperornithinemia and GA in 1974 led to the discovery of the basic enzymic defect, a deficiency of the enzyme ornithine amino transferase (OAT).
Ornithine plays a key role in the urea cycle and most pathways of its metabolism are well known [Figure:1]. OAT is a pyridoxal phosphate-requiring mitochondrial transaminase that catalyzes the reversible interconversion of ornithine and alpha ketoglutarate to pyrroline-5-carboxylate (P5C) and glutamate. The equilibrium is determined by the concentratations of the reacting species in various tissues. OAT has been purified to homogeneity and sequenced from various tissues including the human liver. The defect in OAT activity is expressed in many tissues and cell types from GA patients, including cultured skin fibroblast.[4-5]
As new cases of GA were reported, its clinical heterogeneity became more apparent. It is now best explained in terms of alterations in the OAT gene sequences and the amount of OAT protein detectable in the fibroblast of GA patients. Clinical heterogeneity has been documented with respect to responsiveness to Vitamin B6 therapy, rate of progression of blindness, and expressions of the variable amounts of OAT protein produced by each patient.[6-8]
It is now generally accepted that the demonstration of hyperornithinemia is a prerequisite in clinical cases of GA for the final diagnosis and proper classification of the disease. However, just as there is clinical heterogeneity, the chemical pathology is likely to vary too. This is because cases with GA without accompanying hyperornithinemia have been found, as have cases with enzyme deficit which do not have GA. So it is accepted that chorioretinal degeneration may be due to the toxicity of the accumulated ornithine and/or accumulation or deficiency of polyamines which are metabolites of ornithine and lysine.
Polyamines are formed by the action of ornithine decarboxylase and have deleterious effects on DNA and proteins. They are also reported to affect retinal function. Hence, in addition to ornithine and OAT, polyamines such as putrescine, spermine, spermidine, and cadaverine were also estimated individually in our present work. This article highlights the importance of estimating polyamines in gyrate atrophy.
Materials and Methods
Ornithine assay in plasma
Ornithine was estimated by two-dimensional paper chromatography of plasma treated with sulfosalicyclic acid (1:1). The solvents were n-butanol, acetic acid and water (4:1:5) in the first dimension and pyridine, isoamyl alcohol, and water (10:10:7) in the second dimension. Separated amino acids were stained with ninhydrin. The coloured ornithine spot was extracted with 50% alcohol and assayed spectrophotometrically at 560 nm.
OAT assay in lymphocytes
The lymphocyte cultures were carried out according to Hayasaka's protocol. Cell extracts of phytohaemagglutinin-transformed lymphocytes were prepared by the method of Berger. The enzyme assay was done spectrophotometrically by the method of Katsunuma et al using ornithine as substrate. Protein estimation was done by the method of Lowry et al using BSA as standard. One unit of enzyme activity is equal to that amount of enzyme which releases μM of pyrroline 5-carboxylic acid per mg protein.
Assay of blood and urinary polyamines by HPLC
A 2 ml aliquot of blood or 24-hour urine sample, as the case may be, was hydrolysed in 2 ml of 12 N HCl for 14-16 hr at 100-120°C. After hydrolysis, the sample was adjusted to pH 9.0 and the amines were extracted in n-butanol. The butanol extract was evaporated to dryness and the residue dissolved in 0.1N HCL After the extraction of polyamines, they were benzoylated. For this, benzoylchloride was added to 200μl of. the polyamine aliquots and vortexed for 30 seconds. After 20-minute incubation at 25°C, 2 ml of saturated NaCl was added to the sample to stop the reaction. The benzoyl-polyamines were extracted in 3 ml of diethyl ether (stabilized with about 7 ppm of 2,6, di-tert-butyl 1-4-methyl phenol). The samples were centrifuged at 3000g for 5 minutes and the ether phase was collected and evaporated to dryness over a water bath (60°C). The benzoyl polyamines were redissolved and 200μl of this extract was injected into the HPLC column and chromatographed at 25°C. The elution flow rate was 200μl/ min and the polyamines were detected at 254 nm with an ultraviolet detector using ODS G 18 column; methanol 63% was used as solvent.
A 27-year-old male patient was admitted to Sankara Nethralaya during July 1993 with complaints of diminished night vision since the age of 8 years. This had been progressive, and had been followed by difficulty in day vision also. He also gave a history of decreased field vision in both the eyes and mild hearing difficulty for the last 3 years. He was born of a consanguineous marriage.
He was myopic (-4.00D in the right eye and -2.75D in the left eye). He had anterior subcapsular cataract in both eyes. Fundus examination revealed pale disks in both eyes with arteriolar attenuation. There were tongue-shaped lesions of chorioretinal atrophy with advancing and confluent edges sparing the macula, typical of GA in both eyes. There were pigmented areas due to confluence of the tongue-shaped lesions. ERG showed extinguished responses in both the eyes for photopic and scotopic phases. Field examination with Goldman perimeter revealed tubular fields with a central 10°C field of vision. Fundus fluorescein angiography (FFA) was consistent with GA [Figure:2]. Plasma ornithine levels were 750 μM, OAT in lymphocytes 0.13 μM of pyrroline-5-carboxylic acid/mg protein, and urinary polyamines 72 mg/mg creatinine.
A 15-year-old female patient was examined for gradual loss of vision in both the eyes. She had an episode of fever during her 12th year, associated with convulsions. Her parents' marriage was a case of first-degree consanguinity. Her visual acuity was 1/60 in the right eye and 2/60 in the left eye. The lens and cornea were clear. Fundus examination revealed pale discs, attenuated vessels and unhealthy retinal texture with scalloped margins typical of GA [Figure:3]. Plasma ornithine levels were 1090 μM, OAT in lymphocytes 0.2 μM of pyrroline-5-carboxylic acid / mg protein, and urinary polyamines: 12.3 mg / mg creatinine. Both parents were also investigated clinically and biochemically. They were found to have normal fundus but their plasma ornithine level was elevated.
A 15-year-old boy had pigments in the post-equatorial region. He had no history of night-blindness but a family history of retinitis pigmentosa (RP). Two of his maternal great uncles had severe typical RP. This boy had extinguished ERG and patches of pigmentation. After clinical and laboratory investigations, he was diagnosed with atypical RP [Figure:4]. Interestingly, his mother and brothers had normal ornithine and OAT levels while he had elevated ornithine and lowered OAT levels. Plasma ornithine levels were 568 μM, OAT in lymphocytes 0.06 μM of pyrroline-5-carboxylic acid / mg protein, and urinary polyamines: 63 mg / mg creatinine. His father also had elevated ornithine and lowered OAT levels. All the three members of the family other than the patient had normal fundus.
A 54-year-old female patient was examined with complaints of gradual diminution of vision in both eyes. Her best-corrected visual acuity was 6/24 in the right eye and 6/12 in the left eye. Slitlamp examination revealed immature cataract. Fundus examination showed evidence of disc pallor with lamellar macular hole in both the eyes [Figure:5]. There was also evidence of chorioretinal atrophy involving the inferior hemisphere of the retina suggestive of GA. Although ERG showed normal responses, the plasma ornithine was elevated. Plasma ornithine levels were 696μM, OAT in lymphocytes 0.18 μM of pyrroline-5-carboxylic acid / mg protein, and urinary polyamines: 23.9 mg / mg creatinine. Her younger sister was found to have a normal eye, but elevated ornithine.
A 75-year-old female patient was examined for complaints of gradual painless decrease in vision. Her best-corrected visual acuity was 2/60 in the right eye and the left eye had no vision. Slitlamp examination revealed posterior and anterior subcapsular and nuclear cataracts. Fundus examination showed GA-like changes in the retina. Her plasma ornithine level was elevated. Plasma ornithine levels were 530μM, OAT in lymphocytes 0.07 μM of pyrroline 5 carboxylic acid / mg protein, and urinary polyamines: 58 mg / mg creatinine.
A 37-year-old female patient was examined at Sankara Nethralaya in February 1995 with complaints of defective vision in the right eye since childhood. She was myopic (-9.00 DS) in both eyes and her vision was 6 / 60, N 36 in right eye and 6 / 36, N12 in left eye. Slitlamp examination showed posterior subcapsular cataract in both eyes. Fundus examination showed typical features of GA in both the eyes [Figure:6]. Her ERG showed extinguished responses in both the eyes. Her plasma ornithine levels were 150μM, OAT in lymphocytes 0.4 μM of pyrroline - 5-carboxylic acid /mg protein, but urinary polyamines were 45.7 mg / mg creatinine.
A 50-year-old male patient complained of gradual decrease in vision in both eyes but no night blindness. Slitlamp examination of the anterior segment was unremarkable in both eyes except for a sluggish pupillary defect in the right eye. Fundus examination showed advanced chorioretinal atrophy in both the eyes more pronounced the right eye, with optic atrophy and macular degeneration. Clinical features were suggestive of GA [Figure:7]. There was peripheral pigmentation and attenuation of vessels in both the eyes. His electroretinography showed extinguished responses in both eyes in the scotopic and photopic phases. Plasma ornithine levels were 100 μM, while OAT in lymphocytes was 1.6 μM of pyrroline-5-carboxylic acid / mg protein. Interestingly, the urinary polyamines in this case also were elevated to 63.3 mg / mg creatinine.
The first five cases investigated showed increased plasma ornithine and decreased OAT [Table:1], with statistically significant values. The increase of ornithine was nearly 7-fold while in classical GA it is 10- to 20-fold. However, there was a statistically significant increase in total polyamines, putrescine, spermine.and spermidine, that is 45.8 mg / mg creatinine as against 8 mg / mg creatinine in the urine of the normals [Table:1]. But in cases 6 and 7, plasma ornithine was not significantly elevated; OAT in one case was not significantly reduced. However the polyamines were elevated, that is 45.7 and 63.3 mg / mg creatinine. [Table:1]. HPLC gave a good resolution of individual polyamines [Figure:8]. Individual values of polyamines from ornithine and lysine are given in [Table:2]. There is an increase of putrescine in 6 out of 7 cases and an increase of spermine in 3 patients. It appears that the putrescine level is increased in many cases of GA. In a few cases other than the seven reported above, putrescine was estimated in blood. It was 5±0.3mg in normals, but 80.5±9.6 in patients with GA.
The results are opposite regarding cadaverine. In all 7 cases there is a decrease in the level of this polyamine.
This is expected, as the parent amino acid lysine has been reported to decrease in GA.
From our observation it appears that ornithine levels alone may not be responsible for the disease. This is because in cases 6 and 7, whose clinical features were that of GA, the patients had normal or slightly elevated ornithine and slightly decreased OAT (1.6 in case 8). But polyamines in urine were higher in. both the cases, indicating that polyamines have greater role in GA than ornithine. In respect to other 5 cases also urinary polyamines were found to increase about six-fold. The role of hyperornithinemia is not dismissed, but what is stressed is the role of polyamines and chiefly putrescine, a metabolite of ornithine in GA. In this connection it may be of interest to consider the following observations in the literature.
When mice and chicks were given 5-fluro methylornithine, a selective inactivator of OAT over extended periods of time, they had elevated concentrations of ornithine. But ophthalmoscopic findings of the eye and electroretinography were normal. No toxic effects were seen in either of the animals. From this report it is clear that ornithine is not directly involved in GA.
Low protein (20-35g) and low arginine diet (as arginine is the precursor for ornithine) and B6 (300 mg / day) were tried in three patients. Although they maintained their plasma ornithine concentrations in the range 30-60% of pretherapeutic levels for about 2 years, no significant improvement was noted in the vision or retinal changes. If hyperornithinemia were to be causing the disease, the results should have been different.
Although the association of high levels of ornithine and GA coincide in many cases, increased levels of ornithine alone do not necessarily lead to this degeneration. A patient with known hyperammonemia and a 10-fold increase in plasma ornithine was found to have a normal fundus appearance and normal electroretinogram. There are cases of GA without hyperornithinemia and cases of hyperornithinemia without GA.[17,18]
Recently Wang et al have shown that OAT-deficient mice exhibited neonatal hypo-ornithinemia, and postweaning, they developed hyperornithinemia similar to human GA patients. Subsequent studies in one human GA infant also showed transient hypo-ornithinemia. Thus the OAT reaction appears to play opposite roles in neonatal and adult mammals.
In a case of GA, excretion of glycine, proline and hydroxyproline has also been reported and OAT activity was normal in fibroblasts. In one of our cases too, there was excretion of proline and hydroxyproline. In another report, the ocular disease is reported to progress in juvenile patients despite normal or near normal plasma ornithine concentration.
The condition of a 45-year-old woman with GA was followed up for 12 years and reported. She had ornithinemia, along with massive cysteinemia, massive cystinuria with serum ornithine 740μm / l. These three acids share the same renal mechanism.
It is customary to analyze polyamines in the urine of cancer patients, as they have a low renal threshold. However, it was of interest to assay putrescine in the blood of patients with GA. As given in the results section, blood polyamine level (putrescine) also had increased in the cases investigated.
It appears that more than ornithine, polyamines, the catabolic products of ornithine may be the causative factor, especially because ornithine is an innocuous amino acid. The liver, where it is formed continuously, is not affected. Ornithine decarboxylase has both ornithine and lysine as substrates. In the case of a deficiency of OAT, ornithine decarboxylase may degrade ornithine to polyamines. The higher the concentration of ornithine, the greater the levels of polyamines, namely, putrescine, spermine and spermidine. If the decarboxylase has high Km (Michaelis Menten Constant) for ornithine, it would degrade ornithine with the formation of polyamines. Among the three polyamines putrescine, spermine and spermidine, putrescine appears to be increased significantly in a majority of cases.
Again, it is shown that low levels of lysine could be associated with GA. The polyamine formed from lysine is cadaverine. A low level of lysine might reflect on cadaverine, and this was actually found: while, in the normals the cadaverine was 260 ± 38 mg / mg creatinine, it was 0.94 ± 0.05 in GA patients [Table:1].
In GA, B6 therapy should be done with caution as B6 would activate ornithine decarboxylase also (in addition to OAT) and result in the production of increased polyamines.
In short, emphasis should be directed on measuring polyamines rather than just ornithine in GA because of the following reasons.
1. Polyamines can affect DNA, proteins and enzymes while ornithine does not;
2. They destroy cGMP in the rods of the retina;
3. They are needed for the maturation of the rods and
4. Spermine and spermidine inhibit OAT.
Therefore, in all cases of GA, its desirable to estimate plasma ornithine, OAT as well as polyamines, chiefly putrescine and cadaverine, in order to arrive at a correct diagnosis.
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