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Enzymatic determination of l -ornithine in serum
Climtu
Elsevier
Chimiw
Acfu,
Biomedical
237
121 (1982) 237-243
Press
CCA 2124
Enzymatic Kaarina
determination Ojala
a**, Theodor
” Aurora (Received
H. Weber
und h Kioelti
September
of L-ornithine Hospit&
a and
Helsrnki
Kirsti
in serum K. Takki’
(F~nhd)
16th, 1981: revision January
28th. 1982)
Summary was determined enzymatically using ornithine Serum r_- ornithine carbamoyltransferase (OCT) from Streptococcus faecalis and measuring the citrulline formed with a diacetyl-thiosemicarbazide reaction after precipitation of proteins with trichloracetic acid. The normal fasting values, 26-92 pmol/l, correlated well with those obtained by chromatographic methods.
Introduction The quantitative determination of L-ornithine in serum is needed for diagnosis of and screening or follow-up studies of states with disturbed L-ornithine metabolism. Amino acids are usually analyzed by gas liquid (GLC) or ion exchange (IEX) chromatography. Such methods are rather complicated and time consuming, and expensive equipment is needed. A simpler and more rapid method is therefore useful. The enzymatic method, published recently by Matsuzawa et al [I] is not suitable, because the coupling enzyme is not commercially available. The present paper describes a simple enzymatic method using ornithine carbamoyltransferase as a coupling enzyme: OCT
Carbamoyl
phosphate
+ L-ornithine
The citrulline formed is determined in any clinical laboratory.
+ L-citrulline
+ H3 PO,.
by a color reaction
[2] and can be measured
Materials and methods For enzymatic analysis fasting serum samples and for ion exchange chromatography heparinized plasma, precipitated with sulfosalicylic acid, were used. The samples were stored at -20°C if not analyzed immediately. * Correspondence
to Kaarina
0009.8981/82/oooO-0000/$02.75
Ojala, Clinical
Lab.. Aurora
Hospital,
c 1982 Elsevier Biomedical
Press
SF-00250
Helsinki
25, Finland
Ion exchange chromatography Amino Acid Analyzer.
of amino
acids was performed
with a Technicon
Reagents (1) Tris buffer 0.01 mol pH 8.5. (2) Urease solution (Sigma Chemical Co., St. Louis MD, USA). Dissolve 1 mg ol urease in 3 ml of Tris buffer before use. (3) Dilithium carbamoyl phosphate (Sigma Chemical Co.) 44 mmoljl. Dissolve 10 mg in 1.5 ml of the urease solution immediately before use. (4) Ornithine carbamoyl transferase from Streptococcus fueculis (Sigma Chemical Co), 50 U/l. Dissolve 300 mg in 6 ml of Tris buffer. The solution is divided into 0.5 aliquots and stored at -20°C. (5)~-ornithine standard 500 pmol/l. Stock solution 10 mmol/l: 85 mg of L-ornithine . HCl (Sigma Chemical Co) is dissolved in 50 ml of Tris buffer. Working standard: Stock solution is diluted 1 : 20 with Tris buffer, divided into 1 ml aliquots and stored at -20°C. (6) Acid ferric solution. To 510 ml of distilled water. add 450 ml of concentrated phosphoric acid. 40 ml of concentrated sulfuric acid and 400 mg of ferric chloride. The solution is stable at room temperature for several months. (7) Diacetylmonoxime (DAMO)-thiosemicarbazide (TSC) solution. To 100 ml of distilled water add 500 mg of diacetylmonoxime and 3 mg of thiosemicarbazide. The solution is stable at 4°C in a brown bottle for two months. (8) Color reagents. To 100 ml of the acid ferric solution (Reagent 6) add 20 ml ot DAMO-TSC solution (Reagent 7) before use. (9) Trichloracetic acid (TCA) 10%. Method The assay was performed in Eppendorff micro tubes according to the scheme in Table I. The reaction is started by the addition of OCT. After mixing, the tubes are incubated at 37°C for 30 min. After incubation, 200 ~1 of TCA (Reagent 9) is added to all tubes. After mixing the tubes are centrifuged for 2 min. 200 ~1 of the supernatants are pipetted into test tubes and 3 ml of color reagent added. After mixing the tubes are heated for 15 min in a boiling water bath. The tubes are cooled with running tap water for about 5 min. The absorbances were measured against the blank at a wave length of 5 15 nm on a Turner 380 spectrophotometer. Calculation of the results The following formula
is used (A = absorbance):
( 4aqle - Lnple,O) - ( AreagRI - 4eagl3,O) x 5oo
(4, -4,“)
- PrcagBI - 4eagM,d
pmo,,l
TABLE
I
THE PROCEDURE
FOR L-ORNITHINE
Buffer
Blank Reag. Bl/O Reag. BI St/0 St Sample/O Sample
300 l.ll 200 /&I 200~1 loo /ll loo /II loo/J1 * loo~l*
* In low (normal) buffer.
DETERMINATION
Standard
Sample
_ _
_ _ _ _ _
loo PI _
loo~l* loo~l*
loo PI _
loo /ll la, /ll _
concentrations
of L-ornithine.
Urease Reag. 2
Li ,-carbamoyl-PO,. Urease Reag. 3
_
_ _
IO0 /_ll
OCT Reag. 4
20 20 20 20 20 20
loo PI 100 /Jl _ 100 PI
200 ~1 of the sample can be used without
PI PI /ll /Ll PI fil
addition
of the
Results
Linearity and sensitivity The method is linear
up to 1200 pmol/l
with a sensitivity
of about
10 ~mol/l.
Effect of incubation time Since urea interferes with the color developed by citrulline, urease treatment of samples is necessary [2]. In Fig. 1 the effect of incubation time is presented. The interference of urea has totally disappeared after 20 min incubation as can be seen from the sample blank curve and the production of the citrulline is completed after 20-30 min as seen from the standard. The procedure is capable of removing urea from hyperuricemic sera containing up to 20 mmol/l urea.
0
10
Fig. I. Influence of incubation sample blank (0 -0).
30
time on ornithine
50
values;
sample
(O-
In,”
0).
standard
( n __
n ).
240
Effect of the sample volume When various amounts of serum ranging from 50 to 200 ~1 were tested. there was a linear relationship between the amount of serum and the citrulline formed. Recovery The recovery of L-ornithine added to normal sera was 96- 110%. The recovery was also tested by adding different amounts of L-ornithine to normal sera and comparing the calibration curve obtained with those made for L-ornithine and citrulline. The results can be seen in Fig. 2. Specificity In order to measure basal citrulline, a sample blank made without carbamoyl phosphate is always subtracted from the citrulline formed. Urease is incorporated in the medium to decompose any interfering urea in the samples and reagents. According to Marshall and Cohen [3] OCT from Streptococcus fueculis is specific for t_-ornithine if measured at pH 8.5. Ohshita et al [2] stated that the color reaction can be used without deproteinization when used for the determination of OCT in serum. In our method, however. considerably higher values for L-ornithine are obtained without deproteinization.
AA
0
500
1000 pmolll
Fig. 2. Standard curves for L-omithine, t.-citrulline and semm with serum+ L-omithine (0 -00) W), L-omithine (0 -0). (W -
L-omithine
added:
t.-cltrulline
241
Reproducibility The intra-assay precision different serum samples: mean 97 pmol/l,
of the method
SD i 7.7 ~mol/l,
mean 641 pmol/l,
SD-t
was calculated
by assaying
20 times two
CV 8.0%;
17.2 pmol/l,
CV 2.7%.
The inter-assay precision of the assay, expressed 3.1~ I 1.5% for frozen serum samples.
as a coefficient
of variation,
was
Correlution A comparison between the values obtained with the present method with those obtained with an amino acid analyzer were made. The results are presented in Fig. 3. The values obtained by the two methods are in good agreement: the correlation coefficient is 0.99 and the regression line y = 16.5 + 0.94~. The small differences between methods might be due to the different treatment of the samples used: for enzymatic analysis serum and for amino acid analyzer SSA precipitated plasma samples were used. Serum was used for the enzymatic assay in order to avoid possible interference from added anticoagulants. However, heparinized plasma gave identical results with serum in the enzymatic assay. The amino acid analyzer samples were collected according to the instructions for the instrument. Normal values 21 serum samples obtained from apparently healthy fasting adults were analyzed. The range was 18-106 pmol/l. the calculated normal values (percentiles 2.5-97.5) 26-92 pmol,/l with a mean of 43 pmol/l. The values correlated well with those measured by other methods, as is shown in Table II.
100
0
TechnIcOn
Fig. 3. Correlation Analyzer. rz0.99
800 Aminoacld
1200
pmolll
Analyzer
between results obtained (J’= 16.510.94.~).
with the
present method and with the Technicon
Amino Acid
242 TABLE
II
VENOUS
I.-ORNITHINE
CONCENTRATIONS Number
Prexnt method Ref. No. 4
Sample
(pmol,/l)
IN FASTIN<; Mcthod
ADULTS Rc\ult\
21 32
5
IX
6
20
7
X
I
12
* Gas liquid chromatography. ** Ion e:.-hang chromatography
Discussion It is now well established that patients with gyrate atrophy of the choroid and retina (GA) have a deficiency of the enzyme ornithine-delta-aminotransferase (OAT), with a significant elevation of ornithine in plasma, spinal fluid, aqueous humour and urine [9,10]. These metabolic abnormalities are accompanied by severe and extensive progressive chorioretinal degeneration and cataract formation, but additional clinical symptoms have been either minimal or absent despite significant histologic abnormalities in muscle, hair [l I] and spinal cord [12], indicating metabolic abnormalities also in tissues. Ophthalmic improvement has been recorded in selected cases using arginine-restricted diet for treatment [ 131. The aim of the present investigation was to find a simple but reliable method for the determination of t.-ornithine in serum. We have used the assay for diagnosis of hyperornithinemia in six patients with GA. The values of serum ornithine for these patients .were between 900-1350 ~mol/l. We have also measured t-ornithine in patients with GA on arginine-restricted diet. and values between 360-780 ~mol,/I were found. The proposed method is suitable for the hospital laboratory. It is easy to perform, specific for t_-ornithine and sensitive enough to measure concentrations at the normal level. Acknowledgements Our thanks are to Director V.N. Reddy at the Institute of Biological Sciences, University of Oakland, Rochester, MI, USA, for performing the amino acid analyzer analyses. This study was supported by grants from the Sigrid Juselius Foundation, Finland and NEW Grant EY 00484.
243
References Matsuzawa T, Ito M, Ishiguro I. Enzymatic assays of L-ornithine and L-I-pyrroline-5-carboxylate in tissues, and omithine-load test in human subjects. Anal Biochem 1980; 106: 1-6. Ohshita M. Takeda H, Kamiyama Y. Ozawa K, Honjo I. A direct method for the estimation of ornithine carbamoyltransferase activity in serum. Clin Chim Acta 1976: 67: 145- 152. Marshall M, Cohen P. Omithine transcarbamylase from Srre+coccusfoecu/rs and bovine liver. J Biol Chem 1972; 247: 1654-1668. Adams RF. Determination of amino acid profiles in biological samples by gas chromatography. J Chromatogr 1974; 95: 189-2 12. Lewis A, Waterhouse C. Jacobs LS. Whole-blood and plasma amino acid analysis: gas-liquid and cation-exchange chromatography compared. Clin Chem 1980; 26: 27 I-276. Perry T, Hansen S. Technical pitfalls leading to errors in the quantitation of plasma amino acids. Clin Chim Acta 1969; 25: 53-58. Aoki T’T, Brennan MF, Muller WA, Cahill GF. Jr. Amino acid levels across normal forearm muscle: Whole blood vs. plasma. Adv Enzyme Regul 1974; 14: l57- 165. Matsuwaga T, Ishiguro I. Hyperornithinemia with gyrate atrophy and enzymes involved in ornithinc metabolism of the eye. Biochem Intern 1980; I : 179- 184. 9 Takki K, Simell 0. Genetic aspects in gyrate atrophy of the choroid and retina with hyperornithinaemia. Br J Ophthalmol 1974; 5X: 903-923. Valle D, Kaiser-Kupfer MI, Del Valle LA. Gyrate atrophy of the choroid and retina: deficiency of ornithine aminotransferase in transformed lymphocytes. Proc Nat1 Acad Sci USA 1977; 74: 5 159-5 I6 1. Kaiser-Kupfer MI, Kuwabara T, Askanas V, Brody L, Takki K, Dvoretzky I. Engel K. Systemic manifestations of gyrate atrophy of the choroid and retina. Ophthalmology 19Rl; 8X: 302-306. Takki K. Kuwabara T, Kaiser-Kupfer MI, Sumuvuori H. Histologiska fynd vid atrophia gyrata chorioideae et retinae med hyperornithinaemia (GA). XXV Nordiska Oftalmologmotet, Abstr. 10. 1981. Kaiser-Kupfer MI. Monasterio F, Valle D, Walser M. Brusilow S. Visual results of a long-term trial of a lowarginine diet in gyrate atrophy of choroid and retina. Ophthalmology 19XI ; X8: 307-3 10.
Elsevier
Chimiw
Acfu,
Biomedical
237
121 (1982) 237-243
Press
CCA 2124
Enzymatic Kaarina
determination Ojala
a**, Theodor
” Aurora (Received
H. Weber
und h Kioelti
September
of L-ornithine Hospit&
a and
Helsrnki
Kirsti
in serum K. Takki’
(F~nhd)
16th, 1981: revision January
28th. 1982)
Summary was determined enzymatically using ornithine Serum r_- ornithine carbamoyltransferase (OCT) from Streptococcus faecalis and measuring the citrulline formed with a diacetyl-thiosemicarbazide reaction after precipitation of proteins with trichloracetic acid. The normal fasting values, 26-92 pmol/l, correlated well with those obtained by chromatographic methods.
Introduction The quantitative determination of L-ornithine in serum is needed for diagnosis of and screening or follow-up studies of states with disturbed L-ornithine metabolism. Amino acids are usually analyzed by gas liquid (GLC) or ion exchange (IEX) chromatography. Such methods are rather complicated and time consuming, and expensive equipment is needed. A simpler and more rapid method is therefore useful. The enzymatic method, published recently by Matsuzawa et al [I] is not suitable, because the coupling enzyme is not commercially available. The present paper describes a simple enzymatic method using ornithine carbamoyltransferase as a coupling enzyme: OCT
Carbamoyl
phosphate
+ L-ornithine
The citrulline formed is determined in any clinical laboratory.
+ L-citrulline
+ H3 PO,.
by a color reaction
[2] and can be measured
Materials and methods For enzymatic analysis fasting serum samples and for ion exchange chromatography heparinized plasma, precipitated with sulfosalicylic acid, were used. The samples were stored at -20°C if not analyzed immediately. * Correspondence
to Kaarina
0009.8981/82/oooO-0000/$02.75
Ojala, Clinical
Lab.. Aurora
Hospital,
c 1982 Elsevier Biomedical
Press
SF-00250
Helsinki
25, Finland
Ion exchange chromatography Amino Acid Analyzer.
of amino
acids was performed
with a Technicon
Reagents (1) Tris buffer 0.01 mol pH 8.5. (2) Urease solution (Sigma Chemical Co., St. Louis MD, USA). Dissolve 1 mg ol urease in 3 ml of Tris buffer before use. (3) Dilithium carbamoyl phosphate (Sigma Chemical Co.) 44 mmoljl. Dissolve 10 mg in 1.5 ml of the urease solution immediately before use. (4) Ornithine carbamoyl transferase from Streptococcus fueculis (Sigma Chemical Co), 50 U/l. Dissolve 300 mg in 6 ml of Tris buffer. The solution is divided into 0.5 aliquots and stored at -20°C. (5)~-ornithine standard 500 pmol/l. Stock solution 10 mmol/l: 85 mg of L-ornithine . HCl (Sigma Chemical Co) is dissolved in 50 ml of Tris buffer. Working standard: Stock solution is diluted 1 : 20 with Tris buffer, divided into 1 ml aliquots and stored at -20°C. (6) Acid ferric solution. To 510 ml of distilled water. add 450 ml of concentrated phosphoric acid. 40 ml of concentrated sulfuric acid and 400 mg of ferric chloride. The solution is stable at room temperature for several months. (7) Diacetylmonoxime (DAMO)-thiosemicarbazide (TSC) solution. To 100 ml of distilled water add 500 mg of diacetylmonoxime and 3 mg of thiosemicarbazide. The solution is stable at 4°C in a brown bottle for two months. (8) Color reagents. To 100 ml of the acid ferric solution (Reagent 6) add 20 ml ot DAMO-TSC solution (Reagent 7) before use. (9) Trichloracetic acid (TCA) 10%. Method The assay was performed in Eppendorff micro tubes according to the scheme in Table I. The reaction is started by the addition of OCT. After mixing, the tubes are incubated at 37°C for 30 min. After incubation, 200 ~1 of TCA (Reagent 9) is added to all tubes. After mixing the tubes are centrifuged for 2 min. 200 ~1 of the supernatants are pipetted into test tubes and 3 ml of color reagent added. After mixing the tubes are heated for 15 min in a boiling water bath. The tubes are cooled with running tap water for about 5 min. The absorbances were measured against the blank at a wave length of 5 15 nm on a Turner 380 spectrophotometer. Calculation of the results The following formula
is used (A = absorbance):
( 4aqle - Lnple,O) - ( AreagRI - 4eagl3,O) x 5oo
(4, -4,“)
- PrcagBI - 4eagM,d
pmo,,l
TABLE
I
THE PROCEDURE
FOR L-ORNITHINE
Buffer
Blank Reag. Bl/O Reag. BI St/0 St Sample/O Sample
300 l.ll 200 /&I 200~1 loo /ll loo /II loo/J1 * loo~l*
* In low (normal) buffer.
DETERMINATION
Standard
Sample
_ _
_ _ _ _ _
loo PI _
loo~l* loo~l*
loo PI _
loo /ll la, /ll _
concentrations
of L-ornithine.
Urease Reag. 2
Li ,-carbamoyl-PO,. Urease Reag. 3
_
_ _
IO0 /_ll
OCT Reag. 4
20 20 20 20 20 20
loo PI 100 /Jl _ 100 PI
200 ~1 of the sample can be used without
PI PI /ll /Ll PI fil
addition
of the
Results
Linearity and sensitivity The method is linear
up to 1200 pmol/l
with a sensitivity
of about
10 ~mol/l.
Effect of incubation time Since urea interferes with the color developed by citrulline, urease treatment of samples is necessary [2]. In Fig. 1 the effect of incubation time is presented. The interference of urea has totally disappeared after 20 min incubation as can be seen from the sample blank curve and the production of the citrulline is completed after 20-30 min as seen from the standard. The procedure is capable of removing urea from hyperuricemic sera containing up to 20 mmol/l urea.
0
10
Fig. I. Influence of incubation sample blank (0 -0).
30
time on ornithine
50
values;
sample
(O-
In,”
0).
standard
( n __
n ).
240
Effect of the sample volume When various amounts of serum ranging from 50 to 200 ~1 were tested. there was a linear relationship between the amount of serum and the citrulline formed. Recovery The recovery of L-ornithine added to normal sera was 96- 110%. The recovery was also tested by adding different amounts of L-ornithine to normal sera and comparing the calibration curve obtained with those made for L-ornithine and citrulline. The results can be seen in Fig. 2. Specificity In order to measure basal citrulline, a sample blank made without carbamoyl phosphate is always subtracted from the citrulline formed. Urease is incorporated in the medium to decompose any interfering urea in the samples and reagents. According to Marshall and Cohen [3] OCT from Streptococcus fueculis is specific for t_-ornithine if measured at pH 8.5. Ohshita et al [2] stated that the color reaction can be used without deproteinization when used for the determination of OCT in serum. In our method, however. considerably higher values for L-ornithine are obtained without deproteinization.
AA
0
500
1000 pmolll
Fig. 2. Standard curves for L-omithine, t.-citrulline and semm with serum+ L-omithine (0 -00) W), L-omithine (0 -0). (W -
L-omithine
added:
t.-cltrulline
241
Reproducibility The intra-assay precision different serum samples: mean 97 pmol/l,
of the method
SD i 7.7 ~mol/l,
mean 641 pmol/l,
SD-t
was calculated
by assaying
20 times two
CV 8.0%;
17.2 pmol/l,
CV 2.7%.
The inter-assay precision of the assay, expressed 3.1~ I 1.5% for frozen serum samples.
as a coefficient
of variation,
was
Correlution A comparison between the values obtained with the present method with those obtained with an amino acid analyzer were made. The results are presented in Fig. 3. The values obtained by the two methods are in good agreement: the correlation coefficient is 0.99 and the regression line y = 16.5 + 0.94~. The small differences between methods might be due to the different treatment of the samples used: for enzymatic analysis serum and for amino acid analyzer SSA precipitated plasma samples were used. Serum was used for the enzymatic assay in order to avoid possible interference from added anticoagulants. However, heparinized plasma gave identical results with serum in the enzymatic assay. The amino acid analyzer samples were collected according to the instructions for the instrument. Normal values 21 serum samples obtained from apparently healthy fasting adults were analyzed. The range was 18-106 pmol/l. the calculated normal values (percentiles 2.5-97.5) 26-92 pmol,/l with a mean of 43 pmol/l. The values correlated well with those measured by other methods, as is shown in Table II.
100
0
TechnIcOn
Fig. 3. Correlation Analyzer. rz0.99
800 Aminoacld
1200
pmolll
Analyzer
between results obtained (J’= 16.510.94.~).
with the
present method and with the Technicon
Amino Acid
242 TABLE
II
VENOUS
I.-ORNITHINE
CONCENTRATIONS Number
Prexnt method Ref. No. 4
Sample
(pmol,/l)
IN FASTIN<; Mcthod
ADULTS Rc\ult\
21 32
5
IX
6
20
7
X
I
12
* Gas liquid chromatography. ** Ion e:.-hang chromatography
Discussion It is now well established that patients with gyrate atrophy of the choroid and retina (GA) have a deficiency of the enzyme ornithine-delta-aminotransferase (OAT), with a significant elevation of ornithine in plasma, spinal fluid, aqueous humour and urine [9,10]. These metabolic abnormalities are accompanied by severe and extensive progressive chorioretinal degeneration and cataract formation, but additional clinical symptoms have been either minimal or absent despite significant histologic abnormalities in muscle, hair [l I] and spinal cord [12], indicating metabolic abnormalities also in tissues. Ophthalmic improvement has been recorded in selected cases using arginine-restricted diet for treatment [ 131. The aim of the present investigation was to find a simple but reliable method for the determination of t.-ornithine in serum. We have used the assay for diagnosis of hyperornithinemia in six patients with GA. The values of serum ornithine for these patients .were between 900-1350 ~mol/l. We have also measured t-ornithine in patients with GA on arginine-restricted diet. and values between 360-780 ~mol,/I were found. The proposed method is suitable for the hospital laboratory. It is easy to perform, specific for t_-ornithine and sensitive enough to measure concentrations at the normal level. Acknowledgements Our thanks are to Director V.N. Reddy at the Institute of Biological Sciences, University of Oakland, Rochester, MI, USA, for performing the amino acid analyzer analyses. This study was supported by grants from the Sigrid Juselius Foundation, Finland and NEW Grant EY 00484.
243
References Matsuzawa T, Ito M, Ishiguro I. Enzymatic assays of L-ornithine and L-I-pyrroline-5-carboxylate in tissues, and omithine-load test in human subjects. Anal Biochem 1980; 106: 1-6. Ohshita M. Takeda H, Kamiyama Y. Ozawa K, Honjo I. A direct method for the estimation of ornithine carbamoyltransferase activity in serum. Clin Chim Acta 1976: 67: 145- 152. Marshall M, Cohen P. Omithine transcarbamylase from Srre+coccusfoecu/rs and bovine liver. J Biol Chem 1972; 247: 1654-1668. Adams RF. Determination of amino acid profiles in biological samples by gas chromatography. J Chromatogr 1974; 95: 189-2 12. Lewis A, Waterhouse C. Jacobs LS. Whole-blood and plasma amino acid analysis: gas-liquid and cation-exchange chromatography compared. Clin Chem 1980; 26: 27 I-276. Perry T, Hansen S. Technical pitfalls leading to errors in the quantitation of plasma amino acids. Clin Chim Acta 1969; 25: 53-58. Aoki T’T, Brennan MF, Muller WA, Cahill GF. Jr. Amino acid levels across normal forearm muscle: Whole blood vs. plasma. Adv Enzyme Regul 1974; 14: l57- 165. Matsuwaga T, Ishiguro I. Hyperornithinemia with gyrate atrophy and enzymes involved in ornithinc metabolism of the eye. Biochem Intern 1980; I : 179- 184. 9 Takki K, Simell 0. Genetic aspects in gyrate atrophy of the choroid and retina with hyperornithinaemia. Br J Ophthalmol 1974; 5X: 903-923. Valle D, Kaiser-Kupfer MI, Del Valle LA. Gyrate atrophy of the choroid and retina: deficiency of ornithine aminotransferase in transformed lymphocytes. Proc Nat1 Acad Sci USA 1977; 74: 5 159-5 I6 1. Kaiser-Kupfer MI, Kuwabara T, Askanas V, Brody L, Takki K, Dvoretzky I. Engel K. Systemic manifestations of gyrate atrophy of the choroid and retina. Ophthalmology 19Rl; 8X: 302-306. Takki K. Kuwabara T, Kaiser-Kupfer MI, Sumuvuori H. Histologiska fynd vid atrophia gyrata chorioideae et retinae med hyperornithinaemia (GA). XXV Nordiska Oftalmologmotet, Abstr. 10. 1981. Kaiser-Kupfer MI. Monasterio F, Valle D, Walser M. Brusilow S. Visual results of a long-term trial of a lowarginine diet in gyrate atrophy of choroid and retina. Ophthalmology 19XI ; X8: 307-3 10.