- Email: [email protected]
Use of toluidine blue for sequential staining of urinary amino acid chromatograms
NOTES
CHROM.
1% 5278
Use of Toluidine
Blue for sequential
staining
of urinary
amino
acid chromato-
grams
Two urine screening tests in current use for detecting inborn errors of metabolism are paper chromatography for amino acids1 and the BERRY spot test for mucopolysaccharides2. These tests are usually run separately, but this note describes a means of combining both tests in a single chromatogram using urine volumes containing known quantities of creatinine. The two advantages of this method are that duplicate spotting of urine is eliminated and the screening for mucopolysaccharides is performed on comparable amounts of urine. The creatinine content of the urine is measured by the Jaff6 reaction; the volume of urine applied to the chromatogram is then calculated from the following equation : Volume
of urine
applied
215 (/tl) = CO.0’
where C = mg creatinine per zoo ml of urine. In this equation, the numerator is derived empirically by us, since it provides chromatograms of good quality under our conditions. The denominator is the same as that used by WOOLI;~ in order to correct for the apparent aminoaciduria of dilute urine. One dimensional chromatography is carried out using O’BRIEN’S systeml, but the staining procedure is modified in the following manner. After development of the ninhydrin stain (Pig. IA), a strip containing the points of origin (Al*- o-2) is cut from the proximal section of the chromatogram and overstained with 0.02 y. Toluidine
ovcrstnincd Fig. t . Sections of clrrotnatogratns of urine stttinecl with (A) ninhyclr in ; (13) nitihyclrin rca .gcnt. Urines a.rc from (I) with Toluiclinc Blue; and (C) ninhyclrin ovcrstainecl with Pauly’s wit :h the ncphrotic. synclrotnc a normal Cliilcl; (2) R patient with I-Iurlcr’s synclrotne; (3) a pnticnt adult; (7) a ancl proteinurin; (.I) a child with histiclinctnia; (5) a cystinuric chi :lcl ; (6) a normal ancl (8) a patient with amino aci iclut *ia, and protcinuria clue to pntient with I-lttrlcr’s synclronic; Lowe’s syndrotnc. J.
Clrvorrrnlogr~., 57 (1971)
165-166
*.
166
NOTES
Blue* as in the direct BERRY spot test (Fig. IB). The middle section of the chromatogram (RF z-16) is overstained with Pauly’s reagent for the histidines and the distal section (& 16-100) with Ehrlich’s reagent for proline, hydroxyproline and otller Ehrlich positive compounds in the usual ‘manner. Since neither proteins nor mucopolysaccharides move from the origin in this system, it is possible to detect the presence of both types of compound by this method. Proteins stain with the initial ninhydrin stain (Fig. IA) and with the conventional Pauly’s overstain (Fig. IC), but are decolorized by the Toluidine Blue. Sulfated mucopolysaccharides stain only with the Toluidine Blue (Fig. IB). Since Pauly’s stain of the origin serves only to confirm the presence of proteinuria, we only use it, to stain the middle section of the chromatogram (Xp z-15) for the histidines. We now have used this modification in over IOOO urine samples without finding any false results. Supported by Grants from the National Collins, Foundation of Oregon.
Foundation,
March of Dimes and the
De$artmevtt of Pediatrics, University of Oregon we&al School, 3181 S. W. Sam Jackson Park Road, PortLand, Ore. 97 201 (U.S.A.)
HELEN NEIL
T. CURTIS R. M. BUIST
I D. O’BRIEN, F. A. IDBOTT AND
D. 0. RODGERSON,Labovatovy Manual of Pediatric 4th Ed., Harper and Row, New York, 1968, pp. 24-35. 2 H. K. BERIZYAND J. SPINANGER,J. Lab. Climb Med., 55 (1960) 136. 3 L. I. WOOLF, Grl. Ormond Sir. J., 2 (195x) 61. Biochemical
Received l
Techniques,
January
Micro-
Sth, 1971
Toluicline Blue, 0.2 g; glacial acetic acid, IO ml; g5 Oh ethanol,
J. Chrornalogr., 57 (1971)
20
ml; water to
IOOO
ml.
rG+166
CHROM. 5276 Paper chromatography
of some aurones
Aurones (z-benzalcoumaranones) represent one’of the minor classes of flavonoid compounds. They were discovered as naturally occurring pigments by GEISSLMANAND HEATON~+. Since that time only a few such compounds have been found in nature. Physical and chemical characteristics of these, compounds have been recorded by GEISS~MANAND HAR~ORNE~, HARBORNE*, and I?ARKAS AND PALLOSE. More recently HUI~IZ and coworl
CHROM.
1% 5278
Use of Toluidine
Blue for sequential
staining
of urinary
amino
acid chromato-
grams
Two urine screening tests in current use for detecting inborn errors of metabolism are paper chromatography for amino acids1 and the BERRY spot test for mucopolysaccharides2. These tests are usually run separately, but this note describes a means of combining both tests in a single chromatogram using urine volumes containing known quantities of creatinine. The two advantages of this method are that duplicate spotting of urine is eliminated and the screening for mucopolysaccharides is performed on comparable amounts of urine. The creatinine content of the urine is measured by the Jaff6 reaction; the volume of urine applied to the chromatogram is then calculated from the following equation : Volume
of urine
applied
215 (/tl) = CO.0’
where C = mg creatinine per zoo ml of urine. In this equation, the numerator is derived empirically by us, since it provides chromatograms of good quality under our conditions. The denominator is the same as that used by WOOLI;~ in order to correct for the apparent aminoaciduria of dilute urine. One dimensional chromatography is carried out using O’BRIEN’S systeml, but the staining procedure is modified in the following manner. After development of the ninhydrin stain (Pig. IA), a strip containing the points of origin (Al*- o-2) is cut from the proximal section of the chromatogram and overstained with 0.02 y. Toluidine
ovcrstnincd Fig. t . Sections of clrrotnatogratns of urine stttinecl with (A) ninhyclr in ; (13) nitihyclrin rca .gcnt. Urines a.rc from (I) with Toluiclinc Blue; and (C) ninhyclrin ovcrstainecl with Pauly’s wit :h the ncphrotic. synclrotnc a normal Cliilcl; (2) R patient with I-Iurlcr’s synclrotne; (3) a pnticnt adult; (7) a ancl proteinurin; (.I) a child with histiclinctnia; (5) a cystinuric chi :lcl ; (6) a normal ancl (8) a patient with amino aci iclut *ia, and protcinuria clue to pntient with I-lttrlcr’s synclronic; Lowe’s syndrotnc. J.
Clrvorrrnlogr~., 57 (1971)
165-166
*.
166
NOTES
Blue* as in the direct BERRY spot test (Fig. IB). The middle section of the chromatogram (RF z-16) is overstained with Pauly’s reagent for the histidines and the distal section (& 16-100) with Ehrlich’s reagent for proline, hydroxyproline and otller Ehrlich positive compounds in the usual ‘manner. Since neither proteins nor mucopolysaccharides move from the origin in this system, it is possible to detect the presence of both types of compound by this method. Proteins stain with the initial ninhydrin stain (Fig. IA) and with the conventional Pauly’s overstain (Fig. IC), but are decolorized by the Toluidine Blue. Sulfated mucopolysaccharides stain only with the Toluidine Blue (Fig. IB). Since Pauly’s stain of the origin serves only to confirm the presence of proteinuria, we only use it, to stain the middle section of the chromatogram (Xp z-15) for the histidines. We now have used this modification in over IOOO urine samples without finding any false results. Supported by Grants from the National Collins, Foundation of Oregon.
Foundation,
March of Dimes and the
De$artmevtt of Pediatrics, University of Oregon we&al School, 3181 S. W. Sam Jackson Park Road, PortLand, Ore. 97 201 (U.S.A.)
HELEN NEIL
T. CURTIS R. M. BUIST
I D. O’BRIEN, F. A. IDBOTT AND
D. 0. RODGERSON,Labovatovy Manual of Pediatric 4th Ed., Harper and Row, New York, 1968, pp. 24-35. 2 H. K. BERIZYAND J. SPINANGER,J. Lab. Climb Med., 55 (1960) 136. 3 L. I. WOOLF, Grl. Ormond Sir. J., 2 (195x) 61. Biochemical
Received l
Techniques,
January
Micro-
Sth, 1971
Toluicline Blue, 0.2 g; glacial acetic acid, IO ml; g5 Oh ethanol,
J. Chrornalogr., 57 (1971)
20
ml; water to
IOOO
ml.
rG+166
CHROM. 5276 Paper chromatography
of some aurones
Aurones (z-benzalcoumaranones) represent one’of the minor classes of flavonoid compounds. They were discovered as naturally occurring pigments by GEISSLMANAND HEATON~+. Since that time only a few such compounds have been found in nature. Physical and chemical characteristics of these, compounds have been recorded by GEISS~MANAND HAR~ORNE~, HARBORNE*, and I?ARKAS AND PALLOSE. More recently HUI~IZ and coworl