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Determination of protein in cerebrospinal fluid sources of error in the Lowry method
CLINIC.1 CHIMICA ACT.4
Determination of protein in cerebrospinal fluid Sources of error in the Lowry method In recent publications]. f RIEDER has dra\m attention to the determination of protein with copper and the phenol reagent, of Folin-Ciocalteu. In fact this Folin-Cu methoda, 4 is much more sensitive than the biuret reaction, but unfortunately it is less specific. RIEDER~ has given special attention to the reagent blank, but he does not mention the USCof a sample blank, consisting of the deprotcinizcd ccrebrospinal fluid. This sample blank normally gives a negligible extra extinctions; but SVENSMARK~ TAl3LE I “PROTEIN
VALUES”
01; “.,RIO”S
SO,.“TIOKS
____-_------
BY
OF DRUGS,
‘THE LOWRY
ESTIMATED
AS CEREBROSPI?;AL
-.
.-..-------~ Sodium saliwlatc ;\cet~+alicvllc acid p-Aminosaiic$c acid Phenacctin Chlorp,romazmc Suliamerazine Sulfaguanidine Sulfadiazine Sulfamlamide Penicilhn SLrcptom>;cin Terramycln Chloramphenicol Er~throm~cin Ascorbic acid Sodium Verona1 Tyrosine * hlcasuremcnts wa\elcngth 750 mp.
p.x.s.)
were made in the Beckman
FLUID
METHOD*
25 mg n/A zj mg I!; 2j
lllg
Illg
-
55 121 7 ‘3 15 7 16 30 I4 16 24 5
‘0
50 mg % zj mg I& 50 mg 7% 50 mg y; 50 m6 % 50 mfz 9: 500 Iqml 50 m6 % 10 mg “1 50 “‘fi 90 IO mg ?b j0
-
55
0 0
$;,
0
50 mg Sb IO mg Y/b
33
DI; spectrophotometer,
with ~-cm cuvcttes,
has noted a varying content of color-producing non-protein substances in ccrebrospinal fluid, probably brought about by drugs, and he concluded that it was necessary to determine the actual amounts of these substances in each sample. \Vith the LOWRY method WC found that in most cases (25 were investigated) the amount of color-producing non-protein substances was cquivalcnt to 6 f 3 mg protcin/Ioo ml (as estimated in the trichloroacetic acid titrate of the cercbrospinal fluid) ; this agrees well with L)AUGHAI)AY cl nl. i. However, in one case this sample blank was much higher. The Folin-Cu method gave a “protein value” of 153 rng:& but the proWin-free filtrate proved to bc equivalent to 67 mg ‘$A,so that the real protein concentration was only 86 mg y/i, which was the value found by the biurct method’. As salicylate was the only drug which this patient had received, we considered the possibility that this substance was responsible for the color produced in the sample blank. (Paper chromatographic analysis of the amino acids in the cerebrospinal fluid Clin. Cktm.
Acta.
j
(rg60) ~55-156
SHORT
156
COMMUNICATIONS
failed to demonstrate abnormal quantities of amino acids such as tyrosine and tryptophan, which are known to bc positive to the Folin-Cu reagent). It can be shown easily that sodium salicylate and acetosalicylic acid cause an intensive positive “protein” reaction in the Folin-Cu method, when a solution of the drug is estimated as cerebrospinal fluid. Table I gives the protein equivalents of these compounds together with a number of other common drugs. The same solutions did not produce any color with the biurct reagent ‘. The Folin-Cu method, which has an extinction about IOO times that of the biuret method, is therefore unreliable if the extinction produced by the deproteinizcd sample is neglected. Extra steps thus introduced may affect the accuracy. Clinical Chemical Laboratory, Haarlem (The Netherlands)
St. Elisabeth’s
Hosfiital,
H. A. zOND,lC G. L. VAN ISOETZELAER
1 H. I’. RIELXII, Clin. Chim. Actu, 3 (1958) 455. * H. 1’. I
33 (19.55) 879. 6 0. SVESSMARK, 52and.J. Clin.l.ab. Invest., IO (1958) jo. ' II. L. ROS+ZNTH.~L .&SD II. I. CKSI)IFF, Clin. Chem., 2 (19j6) 394.
Received
September
zgth, 1959
ASSOUNCEMENT
SYMPOS1I.X
OS DRI:GS
AFFECTING
LIPID
METABOLISM
An international Symposium will be held in Milan, Italy, on June, 2-3-4, 1960 for the purpose of reviewing the present status of biological and clinical research on “drugs affecting lipid mctabolism”. The agenda will include four main subjects: I. Sew data on cholesterol and lipid metabolism (biosynthesis, absorption, site, catabolism, excretion, abnormal pathways). 2. Experimental methods for the evaluation of drugs affecting cholesterol and lipid metabolism (new analytical methods and pharmacological tests). 3. Drugs affecting cholesterol and lipid metabolism in relation to the prevention and treatment of experimental atherosclerosis. 4. Clinical methods and therapeutical significance of drugs affecting cholesterol and lipid metabolism. The Symposium, sponsored by the Institute of Pharmacology of the IJniversity of Milan, will be under the Chairmanship of Prof. E. Trabucchi. Information on participation and presentation of papers can be obtained from Prof. S. Garattini, c/o Institute of I’harmacology, Via A. de1 Sarto 21, Milan, Italy until March 1st. The official languages are Italian and English. Simultaneous translation mill be available. The registration fee is US $ IO.
Clin. Chim.
.Acta, 4 (1959) 699. Sate
en bas de page,
au lieu de: “de P2~i, de p2 ni de T-globulincs”. lire: “de &i, ni de P,-macrogl~)blllinc”.
lignc 2:
Determination of protein in cerebrospinal fluid Sources of error in the Lowry method In recent publications]. f RIEDER has dra\m attention to the determination of protein with copper and the phenol reagent, of Folin-Ciocalteu. In fact this Folin-Cu methoda, 4 is much more sensitive than the biuret reaction, but unfortunately it is less specific. RIEDER~ has given special attention to the reagent blank, but he does not mention the USCof a sample blank, consisting of the deprotcinizcd ccrebrospinal fluid. This sample blank normally gives a negligible extra extinctions; but SVENSMARK~ TAl3LE I “PROTEIN
VALUES”
01; “.,RIO”S
SO,.“TIOKS
____-_------
BY
OF DRUGS,
‘THE LOWRY
ESTIMATED
AS CEREBROSPI?;AL
-.
.-..-------~ Sodium saliwlatc ;\cet~+alicvllc acid p-Aminosaiic$c acid Phenacctin Chlorp,romazmc Suliamerazine Sulfaguanidine Sulfadiazine Sulfamlamide Penicilhn SLrcptom>;cin Terramycln Chloramphenicol Er~throm~cin Ascorbic acid Sodium Verona1 Tyrosine * hlcasuremcnts wa\elcngth 750 mp.
p.x.s.)
were made in the Beckman
FLUID
METHOD*
25 mg n/A zj mg I!; 2j
lllg
Illg
-
55 121 7 ‘3 15 7 16 30 I4 16 24 5
‘0
50 mg % zj mg I& 50 mg 7% 50 mg y; 50 m6 % 50 mfz 9: 500 Iqml 50 m6 % 10 mg “1 50 “‘fi 90 IO mg ?b j0
-
55
0 0
$;,
0
50 mg Sb IO mg Y/b
33
DI; spectrophotometer,
with ~-cm cuvcttes,
has noted a varying content of color-producing non-protein substances in ccrebrospinal fluid, probably brought about by drugs, and he concluded that it was necessary to determine the actual amounts of these substances in each sample. \Vith the LOWRY method WC found that in most cases (25 were investigated) the amount of color-producing non-protein substances was cquivalcnt to 6 f 3 mg protcin/Ioo ml (as estimated in the trichloroacetic acid titrate of the cercbrospinal fluid) ; this agrees well with L)AUGHAI)AY cl nl. i. However, in one case this sample blank was much higher. The Folin-Cu method gave a “protein value” of 153 rng:& but the proWin-free filtrate proved to bc equivalent to 67 mg ‘$A,so that the real protein concentration was only 86 mg y/i, which was the value found by the biurct method’. As salicylate was the only drug which this patient had received, we considered the possibility that this substance was responsible for the color produced in the sample blank. (Paper chromatographic analysis of the amino acids in the cerebrospinal fluid Clin. Cktm.
Acta.
j
(rg60) ~55-156
SHORT
156
COMMUNICATIONS
failed to demonstrate abnormal quantities of amino acids such as tyrosine and tryptophan, which are known to bc positive to the Folin-Cu reagent). It can be shown easily that sodium salicylate and acetosalicylic acid cause an intensive positive “protein” reaction in the Folin-Cu method, when a solution of the drug is estimated as cerebrospinal fluid. Table I gives the protein equivalents of these compounds together with a number of other common drugs. The same solutions did not produce any color with the biurct reagent ‘. The Folin-Cu method, which has an extinction about IOO times that of the biuret method, is therefore unreliable if the extinction produced by the deproteinizcd sample is neglected. Extra steps thus introduced may affect the accuracy. Clinical Chemical Laboratory, Haarlem (The Netherlands)
St. Elisabeth’s
Hosfiital,
H. A. zOND,lC G. L. VAN ISOETZELAER
1 H. I’. RIELXII, Clin. Chim. Actu, 3 (1958) 455. * H. 1’. I
33 (19.55) 879. 6 0. SVESSMARK, 52and.J. Clin.l.ab. Invest., IO (1958) jo. ' II. L. ROS+ZNTH.~L .&SD II. I. CKSI)IFF, Clin. Chem., 2 (19j6) 394.
Received
September
zgth, 1959
ASSOUNCEMENT
SYMPOS1I.X
OS DRI:GS
AFFECTING
LIPID
METABOLISM
An international Symposium will be held in Milan, Italy, on June, 2-3-4, 1960 for the purpose of reviewing the present status of biological and clinical research on “drugs affecting lipid mctabolism”. The agenda will include four main subjects: I. Sew data on cholesterol and lipid metabolism (biosynthesis, absorption, site, catabolism, excretion, abnormal pathways). 2. Experimental methods for the evaluation of drugs affecting cholesterol and lipid metabolism (new analytical methods and pharmacological tests). 3. Drugs affecting cholesterol and lipid metabolism in relation to the prevention and treatment of experimental atherosclerosis. 4. Clinical methods and therapeutical significance of drugs affecting cholesterol and lipid metabolism. The Symposium, sponsored by the Institute of Pharmacology of the IJniversity of Milan, will be under the Chairmanship of Prof. E. Trabucchi. Information on participation and presentation of papers can be obtained from Prof. S. Garattini, c/o Institute of I’harmacology, Via A. de1 Sarto 21, Milan, Italy until March 1st. The official languages are Italian and English. Simultaneous translation mill be available. The registration fee is US $ IO.
Clin. Chim.
.Acta, 4 (1959) 699. Sate
en bas de page,
au lieu de: “de P2~i, de p2 ni de T-globulincs”. lire: “de &i, ni de P,-macrogl~)blllinc”.
lignc 2: