181
Clinica Chimica Acta, 65 (1975) 181-185 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 7348
AN IMPROVED METHOD FOR THE DETERMINATION OF 5-PHOSPHORIBOSYL l-PYROPHOSPHATE
V. MICHELI, Institute
(Received
G. POMPUCCI
of Biochemistry
and R. MARCOLOdGO*
and Cattedra
di Reumatologia,
Universith
di Siena,
Siena
(Italy)
June 2, 1975)
Summary A spectrophotometric method for the determination of 5-phosphoribosyl 1-pyrophosphate (PRPP) is presented which shows several advantages in comparison to the radiochemical techniques, such as a relatively simple, rapid and less expensive procedure. This technique has been used to evaluate PRPP content in erythrocytes, leukocytes and lymphocytes of normal subjects and individuals with partial hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) deficiency. The results obtained proved to be completely reliable in both groups of subjects examined, with values of PRPP similar to those observed by radiochemical techniques.
Introduction Studies in vitro and in vivo have suggested that the intracellular concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP) appears to be important in the regulation of purine biosynthesis de novo [l-6]. PRPP is a limiting substrate in the first reaction of purine synthesis which is catalyzed by glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14), the probable rate-limiting enzyme in this pathway, and is also a substrate in the phosphoribosyltransferase reactions constituting the salvage pathway of purine nucleotides [ 5,7]. The assay of PRPP in human cells such as erythrocytes and fibroblasts has permitted the study of intracellular levels of this substrate under a variety of physiological and pathological conditions. Increased concentrations of PRPP have been observed in subjects with partial or complete absence of hypoxanthine guanine phosphoribosyltransferase [ 5,8,9] , while an increased PRPP formation or turnover have been reported in normal subjects after * Address for correspondence: Via Donizetti I. Siena, Italy.
Dr. R. Marcolongo.
Cattedra di Reumatologia.
University of Siena,
glucsose and fructose load [ 10 J a11(1111somr;’ l)ai,leni,> with !)r1tnarv ~;lrl.ai~cil!: gout [l-3,11] . Since ahnormalitles of PRPE’ synthesis might contnbute to thtl pathogenesis of some cases of hyperuricemia, the st,udy of PRPP concentration and regulation may be helpful to elucidate the mechanisms leading to purine metabolism alterations in gout. The methods for the determination of PRPP described thus far are radiochemical and utilize the incorporation of [I “C] adenine or [’ “C] hypoxanthine into the corresponding nucleotides, adenosinr monophosphate (AMP) and inosine monophosphate (IMP), in the presence of the specific phosphoribosyltransferase enzymes, followed by chromatography to separate the radioactive nucleotides formed in the reaction [l-3,12,13]. However, such methods are very elaborate and expensive, and may he carried out only in well-equipped laboratories, preventing this test from entering into the diagnostic routine of hyperuricemic states. Ke present an improved method for the determination of PRPP that we found to be accurate, sensitive and reproducible, as well as rapid and easy to carry out. Materials and methods PRPP content was measured in erythrocytes, leukocytes and lymphocytes of normal adult subjects, subjects with partial HGPRTase deficiency and in liver and erythrocytes of some mammalian species. The group of subjects used consisted of 10 healthy adult volunteers (mean age 35 years) with serum uric acid concentrations within the normal range (4.12-5.49 mg/lOO ml) and 3 subjects with partial deficiency in hypoxanthine guanine phosphoribosyltransferase and hyperuricemia. Plasma, leukocytes and platelets were removed from freshly drawn heparinized blood by centrifugation and the erythrocytes washed twice with cold saline. Leukocytes were isolated from whole blood according to the procedure of Chodirker et al. [14]. Briefly, 10 ml of venous blood were diluted 20-fold with saline containing bovine serum albumin 0.1%; the suspension was then centrifuged for 30 min at 800 X g and the sediment of leukocytes and erythrocytes resuspended for 20 s in hypotonic solution (NaCl 0.2%) to lyse the erythrocytes, and then an equal volume of hypertonic solution (NaCl 1.61%) was added. The leukocytes were washed with isotonic saline and lysing and washing procedures were then repeated. Leukocytes were counted in the final suspension and their concentration adjusted as necessary. Final leukocyte recovery was about 57% with this procedure. Lymphocytes were isolated from whole blood according to the method of Thorsby and Bratlie [15], employing sedimentation in Hysopaque and Ficoll and successive washing and concentration by centrifugation. Isolated and packed erythrocytes, leukocytes or lymphocytes were resuspended in EDTA 1 mM, pH 7, in order to avoid hydrolysis of PRPP, and the suspensions were heated for 2 min in boiling water and then chilled (4°C) immediately. Precipitated proteins were packed by centrifugation for 15 min at 6000 X g, and the supernatant was collected and mixed with 0.1 ml of Norit A charcoal to absorb endogenous nucleotides. After mixing, the charcoal was removed by centrifugation at 6000 X g for 10 min. A clear supernatant was obtained which was used for the determination of PRPP concentration. PRPP was assayed following the procedure of Kornberg et al. [16] modified in our laboratory. This method is based on the spectrophoto-
metric disappearance of orotate absorbance at 295 nm, and had been used by these authors to estimate PRPP production in vitro or PRPP purity in industrial preparations. We modified the procedure to apply it to the determination of PRPP in crude cellular extracts. Reaction mixtures contained: Tris/HCl buffer, pH 8, 20 pmol; orotate 0.03 pmol; MgCl, 0.6 pmol; PRPP solution or cell extracts containing at least 3-10 pmol. A first spectrophotometric reading was made at 295 nm to determine the initial absorbance. 0.075 Units of orotidine-5’-phosphate pyrophosphorylase (EC 2.4.2.10) and orotidine-5’-phosphate decarboxylase (EC 4.1.1.23) were then added to the mixture and the absorbance decrease was followed at 295 nm for approximately 15-20 min. The reaction scheme is as follows: orotidine + PRPP “r”tidine-5’-ph”sphate~
Orotate
5’-phosphate
+ PP,
Mg2+
Orotidine
5’-phosphate
“r”tidine-5-ph”sphat~uridine 5-phosphate
+ CO,
Mg2+
Concentration values were expressed as nmol/106 cells for leukocytes and lymphocytes and as nmol/g Hb for erythrocytes. Hemoglobin content was determined on erythrocyte lysate before boiling, using a cyanmethemoglobin method. This was preferred to the packed cell volume parameter usually reported in the literature, being more precise and reproducible. PRPP sodium salt, orotidine-5’-phosphate pyrophosphorylase and orotidine-5’-phosphate decarboxylase samples were purchased from Sigma Chemical Co. (St. Louis, U.S.A.) and from Boehringkr (Mannheim, West Germany). Results Table I shows the erythrocyte, leukocyte and lymphocyte PRPP concentrations found in normal subjects and subjects with partial deficiency in hypoxanthine guanine phosphoribosyltransferase. Intracellular PRPP content was within the normal range in erythrocytes, leukocytes and lymphocytes from 10 healthy subjects, but was significantly higher (1.5- to 3-fold the normal values) in erythrocytes and leukocytes from 3 subjects with partial deficiency in hypoxanthine guanine phosphoribosyltransferase. The mean erythrocyte PRPP content was 16 + 9.2 nmol/g Hb in normal subjects and 39 f 1.6 nmol/g Hb in subjects with partial deficiency in hypoxanthine guanine phosphoribosyltrans-
TABLE
I
CONCENTRATION MAL
SUBJECTS
OF AND
PRPP
IN
ERYTHROCYTES.
INDIVIDUALS
WITH
LEUKOCYTES
PARTIAL
AND
DEFICIENCY
LYMPHOCYTES
OF
IN HYPOXANTHINE
NOR-
GUANINE
PHOSPHORIBOSYLTRANSFERASE Erythrocyte
Normal Subjects
subjects with
partial
deficiency
PRPP
Leukocyte
concentration
concentration
(nmol/g
(nmoI/l@
Hb)
PRPP
Lymphocyte
PRPP
concentration cells)
(nmoI/l@
16
f 9.2
0.12
f 0.079
0.11
39
+ 1.6
0.64
? 0.547
Not
cells)
* 0.055 determined
184
ferase. The mean leukocyte PRPP concentration was 0.12 + 0.079 nmol/lO” cells in normal subjects and 0.64 + 0.547 nmol/lO’ cells in hypoxanthine guanine phosphoribosyltransferase deficient subjects, while the lymphocyte PRPP content found in normal subjects was 0.11 t- 0.055 nmol/lO’ cells. Discussion The levels of PRPP measured in erythrocytes with our method agree with the values reported by other authors [l-13]. The sensitivity of the method was tested on increasing concentrations of PRPP standard solutions and was shown to reach 2.5 nmol (Fig. 1). The applicability of the method to tissue extracts was supported by the experiments carried out on rat liver samples (Table II). The recovery of PRPP during the extraction procedure was controlled by boiling and Norit A-treating known standard solutions of PRPP, and by adding a known aliquot of PRPP to cell suspensions before the boiling procedure. This last method was commonly used in order to increase the sensitivity of the technique to detect small PRPP quantities. A linear relationship was obtained between the amount of PRPP added to hemolysate and its recovery, within a concentration of lo-12 pmol/ml (Fig. 2). During heating at lOO”C, PRPP loss varied between O-10%, while treatment with Norit A did not alter significantly PRPP content. The PRPP values obtained in our series of subjects by the spectrophorometric method were controlled by the radiochemical technique, as modified by Sperling et al. [13]. PRPP content was slightly higher than the values reported in the literature with this latter procedure (2.1-7.5 instead of 1.4-4.6 nmol/ml packed erythrocytes, in normal subjects). However, our results agree with those observed by other authors in healthy subjects and individuals with hypoxanthine guanine phosphoribosyltransferase deficiency [3,5,8,9,12,13]. The results obtained with leukocytes and lymphocytes are very interesting and are worthy of further investigation to ascertain whether these cells are capable of de novo purine biosynthesis [ 171 .
Fig. 1. Change in absorbance
as a function
of the PRPP concentration.
Fig. 2. Linear relationship between the amount of PRPP added to the cell suspension, obtained in the assay). a. leukocytes: b. erythrocytes. (expressed as AE,,,
and its recovery
185 TABLE
II
OF PRPP
CONCENTRATION
IN
Erythrocyte
ERYTHROCYTES
AND
PRPP
Liver
LIVER
concentration
(nmol/g
(nmol/g
fresh
COW
126
i 25.2
Not
determined
243
f 56
Not
determined
92
+ 15.1
Not
Rat
123
f 23.5
52
MAMMALIAN
SPECIES
tissue)
Calf Pig
SOME
PRPP
concentration Hb)
0~
determined + 19.9
The advantage of our modified procedure for the determination of PRPP lies in its relative simplicity, avoiding the use of radiochemical reagents; the method is applicable to several kinds of cells and tissue extracts and is simpler and more rapid than radiochemical techniques. The whole procedure should take no longer than 2 h for erythrocytes and lymphocytes and 3 h for leukocytes, while radiochemical methods take much longer, requiring the chromatography of formed nucleotides. The method has the advantage of being suitable for clinical purposes and diagnostic screening, it is not expensive and is simple to carry out, assuring at the same time satisfactory sensitivity and reliability. References 1
Jones,
2
Hershko,A.,
O.W.,
3
Hershko,
4
Greene.
D.M.
Hershko,C. A.,
M.L.
5
Fox,
6
Wyngaarden,
I.H.
and
7
Kelley,
Marcolongo,
9
Gordon,
and
W.N..
Fox,
I.H.
R. and
R.B.,
Marcolongo,
(1969)
(1971)
Ann.
J. Med.
56,
and Wyngaarden,
DeboIini.
A. D.,
J. Clin. Sci.
Biochim.
Int.
Biophys.
Rheumat.
Med.
Invest.
41.
1805-1815
4, 939-944 Acta
184,
64-76
12.666-667
74.424433
651464 (1970)
Schw.
Emmerson. Jappellt,
(1962)
Isr. J. Med.
Arthr.
J.B.
(1974)
L. and
D’AIonzo.
J.B.
J. (1968)
J. (1969)
J.E.
Am.
Thompson, R.,
Mager.
W.N.
(1974)
Wyngaarden,
Mager,
SeegmiUer,
KeIIey. J.B.
and and
RaziqA.
and
8 10
Ashton,
Clin.
Med. B.T.
Res.
18,457
Wochenschr.
(1974)
104.1411-1416
Metabolism
R..
Riario-Sfona,
J.E.
(1973)
Am.
Metabolism
20,
G.
23.
and
921427
BartaIi.
R.
(1975)
Reumatismo,
in press 11
Becker,
12
Meyskens.
13
Sperling.
14
Chodirker,
15
Thorsby,
M.A..
Meyer.
F.L. 0..
L.J.
and
and Williams,
E&m, W.B.,
E. and
G., Bock.
SeegmiIler. H.E.
Persky-Brash, G.N.
Bratlie,
A.
(1971)
S. and De Vries,
and Vaughan. (1970)
J.H.
A.
(1968)
J. Med.
55,
232-242
737-742 (1972)
J. Lab.
in Histocompatibility
J. Lab. Clin.
Testing,
Med.
Clin. 71,
PP. 655-656,
hagen 16
Komberg,
17
Murray,
A., A.W.
Lieberman, (1971)
Ann.
I. and Simms, Rev.
Biochem.
E.S.
(1955)
40,811-826
J. Biol.
Chem.
215,
389401
Med.
79.
1021-1026
9-19 Munksgaard,
Copen-
Clinica Chimica Acta, 65 (1975) 181-185 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 7348
AN IMPROVED METHOD FOR THE DETERMINATION OF 5-PHOSPHORIBOSYL l-PYROPHOSPHATE
V. MICHELI, Institute
(Received
G. POMPUCCI
of Biochemistry
and R. MARCOLOdGO*
and Cattedra
di Reumatologia,
Universith
di Siena,
Siena
(Italy)
June 2, 1975)
Summary A spectrophotometric method for the determination of 5-phosphoribosyl 1-pyrophosphate (PRPP) is presented which shows several advantages in comparison to the radiochemical techniques, such as a relatively simple, rapid and less expensive procedure. This technique has been used to evaluate PRPP content in erythrocytes, leukocytes and lymphocytes of normal subjects and individuals with partial hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) deficiency. The results obtained proved to be completely reliable in both groups of subjects examined, with values of PRPP similar to those observed by radiochemical techniques.
Introduction Studies in vitro and in vivo have suggested that the intracellular concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP) appears to be important in the regulation of purine biosynthesis de novo [l-6]. PRPP is a limiting substrate in the first reaction of purine synthesis which is catalyzed by glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14), the probable rate-limiting enzyme in this pathway, and is also a substrate in the phosphoribosyltransferase reactions constituting the salvage pathway of purine nucleotides [ 5,7]. The assay of PRPP in human cells such as erythrocytes and fibroblasts has permitted the study of intracellular levels of this substrate under a variety of physiological and pathological conditions. Increased concentrations of PRPP have been observed in subjects with partial or complete absence of hypoxanthine guanine phosphoribosyltransferase [ 5,8,9] , while an increased PRPP formation or turnover have been reported in normal subjects after * Address for correspondence: Via Donizetti I. Siena, Italy.
Dr. R. Marcolongo.
Cattedra di Reumatologia.
University of Siena,
glucsose and fructose load [ 10 J a11(1111somr;’ l)ai,leni,> with !)r1tnarv ~;lrl.ai~cil!: gout [l-3,11] . Since ahnormalitles of PRPE’ synthesis might contnbute to thtl pathogenesis of some cases of hyperuricemia, the st,udy of PRPP concentration and regulation may be helpful to elucidate the mechanisms leading to purine metabolism alterations in gout. The methods for the determination of PRPP described thus far are radiochemical and utilize the incorporation of [I “C] adenine or [’ “C] hypoxanthine into the corresponding nucleotides, adenosinr monophosphate (AMP) and inosine monophosphate (IMP), in the presence of the specific phosphoribosyltransferase enzymes, followed by chromatography to separate the radioactive nucleotides formed in the reaction [l-3,12,13]. However, such methods are very elaborate and expensive, and may he carried out only in well-equipped laboratories, preventing this test from entering into the diagnostic routine of hyperuricemic states. Ke present an improved method for the determination of PRPP that we found to be accurate, sensitive and reproducible, as well as rapid and easy to carry out. Materials and methods PRPP content was measured in erythrocytes, leukocytes and lymphocytes of normal adult subjects, subjects with partial HGPRTase deficiency and in liver and erythrocytes of some mammalian species. The group of subjects used consisted of 10 healthy adult volunteers (mean age 35 years) with serum uric acid concentrations within the normal range (4.12-5.49 mg/lOO ml) and 3 subjects with partial deficiency in hypoxanthine guanine phosphoribosyltransferase and hyperuricemia. Plasma, leukocytes and platelets were removed from freshly drawn heparinized blood by centrifugation and the erythrocytes washed twice with cold saline. Leukocytes were isolated from whole blood according to the procedure of Chodirker et al. [14]. Briefly, 10 ml of venous blood were diluted 20-fold with saline containing bovine serum albumin 0.1%; the suspension was then centrifuged for 30 min at 800 X g and the sediment of leukocytes and erythrocytes resuspended for 20 s in hypotonic solution (NaCl 0.2%) to lyse the erythrocytes, and then an equal volume of hypertonic solution (NaCl 1.61%) was added. The leukocytes were washed with isotonic saline and lysing and washing procedures were then repeated. Leukocytes were counted in the final suspension and their concentration adjusted as necessary. Final leukocyte recovery was about 57% with this procedure. Lymphocytes were isolated from whole blood according to the method of Thorsby and Bratlie [15], employing sedimentation in Hysopaque and Ficoll and successive washing and concentration by centrifugation. Isolated and packed erythrocytes, leukocytes or lymphocytes were resuspended in EDTA 1 mM, pH 7, in order to avoid hydrolysis of PRPP, and the suspensions were heated for 2 min in boiling water and then chilled (4°C) immediately. Precipitated proteins were packed by centrifugation for 15 min at 6000 X g, and the supernatant was collected and mixed with 0.1 ml of Norit A charcoal to absorb endogenous nucleotides. After mixing, the charcoal was removed by centrifugation at 6000 X g for 10 min. A clear supernatant was obtained which was used for the determination of PRPP concentration. PRPP was assayed following the procedure of Kornberg et al. [16] modified in our laboratory. This method is based on the spectrophoto-
metric disappearance of orotate absorbance at 295 nm, and had been used by these authors to estimate PRPP production in vitro or PRPP purity in industrial preparations. We modified the procedure to apply it to the determination of PRPP in crude cellular extracts. Reaction mixtures contained: Tris/HCl buffer, pH 8, 20 pmol; orotate 0.03 pmol; MgCl, 0.6 pmol; PRPP solution or cell extracts containing at least 3-10 pmol. A first spectrophotometric reading was made at 295 nm to determine the initial absorbance. 0.075 Units of orotidine-5’-phosphate pyrophosphorylase (EC 2.4.2.10) and orotidine-5’-phosphate decarboxylase (EC 4.1.1.23) were then added to the mixture and the absorbance decrease was followed at 295 nm for approximately 15-20 min. The reaction scheme is as follows: orotidine + PRPP “r”tidine-5’-ph”sphate~
Orotate
5’-phosphate
+ PP,
Mg2+
Orotidine
5’-phosphate
“r”tidine-5-ph”sphat~uridine 5-phosphate
+ CO,
Mg2+
Concentration values were expressed as nmol/106 cells for leukocytes and lymphocytes and as nmol/g Hb for erythrocytes. Hemoglobin content was determined on erythrocyte lysate before boiling, using a cyanmethemoglobin method. This was preferred to the packed cell volume parameter usually reported in the literature, being more precise and reproducible. PRPP sodium salt, orotidine-5’-phosphate pyrophosphorylase and orotidine-5’-phosphate decarboxylase samples were purchased from Sigma Chemical Co. (St. Louis, U.S.A.) and from Boehringkr (Mannheim, West Germany). Results Table I shows the erythrocyte, leukocyte and lymphocyte PRPP concentrations found in normal subjects and subjects with partial deficiency in hypoxanthine guanine phosphoribosyltransferase. Intracellular PRPP content was within the normal range in erythrocytes, leukocytes and lymphocytes from 10 healthy subjects, but was significantly higher (1.5- to 3-fold the normal values) in erythrocytes and leukocytes from 3 subjects with partial deficiency in hypoxanthine guanine phosphoribosyltransferase. The mean erythrocyte PRPP content was 16 + 9.2 nmol/g Hb in normal subjects and 39 f 1.6 nmol/g Hb in subjects with partial deficiency in hypoxanthine guanine phosphoribosyltrans-
TABLE
I
CONCENTRATION MAL
SUBJECTS
OF AND
PRPP
IN
ERYTHROCYTES.
INDIVIDUALS
WITH
LEUKOCYTES
PARTIAL
AND
DEFICIENCY
LYMPHOCYTES
OF
IN HYPOXANTHINE
NOR-
GUANINE
PHOSPHORIBOSYLTRANSFERASE Erythrocyte
Normal Subjects
subjects with
partial
deficiency
PRPP
Leukocyte
concentration
concentration
(nmol/g
(nmoI/l@
Hb)
PRPP
Lymphocyte
PRPP
concentration cells)
(nmoI/l@
16
f 9.2
0.12
f 0.079
0.11
39
+ 1.6
0.64
? 0.547
Not
cells)
* 0.055 determined
184
ferase. The mean leukocyte PRPP concentration was 0.12 + 0.079 nmol/lO” cells in normal subjects and 0.64 + 0.547 nmol/lO’ cells in hypoxanthine guanine phosphoribosyltransferase deficient subjects, while the lymphocyte PRPP content found in normal subjects was 0.11 t- 0.055 nmol/lO’ cells. Discussion The levels of PRPP measured in erythrocytes with our method agree with the values reported by other authors [l-13]. The sensitivity of the method was tested on increasing concentrations of PRPP standard solutions and was shown to reach 2.5 nmol (Fig. 1). The applicability of the method to tissue extracts was supported by the experiments carried out on rat liver samples (Table II). The recovery of PRPP during the extraction procedure was controlled by boiling and Norit A-treating known standard solutions of PRPP, and by adding a known aliquot of PRPP to cell suspensions before the boiling procedure. This last method was commonly used in order to increase the sensitivity of the technique to detect small PRPP quantities. A linear relationship was obtained between the amount of PRPP added to hemolysate and its recovery, within a concentration of lo-12 pmol/ml (Fig. 2). During heating at lOO”C, PRPP loss varied between O-10%, while treatment with Norit A did not alter significantly PRPP content. The PRPP values obtained in our series of subjects by the spectrophorometric method were controlled by the radiochemical technique, as modified by Sperling et al. [13]. PRPP content was slightly higher than the values reported in the literature with this latter procedure (2.1-7.5 instead of 1.4-4.6 nmol/ml packed erythrocytes, in normal subjects). However, our results agree with those observed by other authors in healthy subjects and individuals with hypoxanthine guanine phosphoribosyltransferase deficiency [3,5,8,9,12,13]. The results obtained with leukocytes and lymphocytes are very interesting and are worthy of further investigation to ascertain whether these cells are capable of de novo purine biosynthesis [ 171 .
Fig. 1. Change in absorbance
as a function
of the PRPP concentration.
Fig. 2. Linear relationship between the amount of PRPP added to the cell suspension, obtained in the assay). a. leukocytes: b. erythrocytes. (expressed as AE,,,
and its recovery
185 TABLE
II
OF PRPP
CONCENTRATION
IN
Erythrocyte
ERYTHROCYTES
AND
PRPP
Liver
LIVER
concentration
(nmol/g
(nmol/g
fresh
COW
126
i 25.2
Not
determined
243
f 56
Not
determined
92
+ 15.1
Not
Rat
123
f 23.5
52
MAMMALIAN
SPECIES
tissue)
Calf Pig
SOME
PRPP
concentration Hb)
0~
determined + 19.9
The advantage of our modified procedure for the determination of PRPP lies in its relative simplicity, avoiding the use of radiochemical reagents; the method is applicable to several kinds of cells and tissue extracts and is simpler and more rapid than radiochemical techniques. The whole procedure should take no longer than 2 h for erythrocytes and lymphocytes and 3 h for leukocytes, while radiochemical methods take much longer, requiring the chromatography of formed nucleotides. The method has the advantage of being suitable for clinical purposes and diagnostic screening, it is not expensive and is simple to carry out, assuring at the same time satisfactory sensitivity and reliability. References 1
Jones,
2
Hershko,A.,
O.W.,
3
Hershko,
4
Greene.
D.M.
Hershko,C. A.,
M.L.
5
Fox,
6
Wyngaarden,
I.H.
and
7
Kelley,
Marcolongo,
9
Gordon,
and
W.N..
Fox,
I.H.
R. and
R.B.,
Marcolongo,
(1969)
(1971)
Ann.
J. Med.
56,
and Wyngaarden,
DeboIini.
A. D.,
J. Clin. Sci.
Biochim.
Int.
Biophys.
Rheumat.
Med.
Invest.
41.
1805-1815
4, 939-944 Acta
184,
64-76
12.666-667
74.424433
651464 (1970)
Schw.
Emmerson. Jappellt,
(1962)
Isr. J. Med.
Arthr.
J.B.
(1974)
L. and
D’AIonzo.
J.B.
J. (1968)
J. (1969)
J.E.
Am.
Thompson, R.,
Mager.
W.N.
(1974)
Wyngaarden,
Mager,
SeegmiUer,
KeIIey. J.B.
and and
RaziqA.
and
8 10
Ashton,
Clin.
Med. B.T.
Res.
18,457
Wochenschr.
(1974)
104.1411-1416
Metabolism
R..
Riario-Sfona,
J.E.
(1973)
Am.
Metabolism
20,
G.
23.
and
921427
BartaIi.
R.
(1975)
Reumatismo,
in press 11
Becker,
12
Meyskens.
13
Sperling.
14
Chodirker,
15
Thorsby,
M.A..
Meyer.
F.L. 0..
L.J.
and
and Williams,
E&m, W.B.,
E. and
G., Bock.
SeegmiIler. H.E.
Persky-Brash, G.N.
Bratlie,
A.
(1971)
S. and De Vries,
and Vaughan. (1970)
J.H.
A.
(1968)
J. Med.
55,
232-242
737-742 (1972)
J. Lab.
in Histocompatibility
J. Lab. Clin.
Testing,
Med.
Clin. 71,
PP. 655-656,
hagen 16
Komberg,
17
Murray,
A., A.W.
Lieberman, (1971)
Ann.
I. and Simms, Rev.
Biochem.
E.S.
(1955)
40,811-826
J. Biol.
Chem.
215,
389401
Med.
79.
1021-1026
9-19 Munksgaard,
Copen-
