263
Clinica Chimica Acta, 66 (1976) 263-266 @ Elsevier Scientific Publishing Company,
SHORT
Amsterdam
-
Printed
in The Netherlands
COMMUNICATION ____
CCA 7402
ESTIMATION OF METHYLMALONIC BY THIN-LAYER CHROMATOGRAPHY
K.C. DAS*,
Department Chandigarh (Received
GEETA
SHARMA,
of Haematology, (India)
DIPIKA
ACID
MOHANTY
Postgraduate
IN URINE
and K.L. CHACHRA
Institute
of Medical
Education
and Research,
July 2, 1975)
Summary We describe a relatively simple method for the quantitative estimation of methylmalonic acid in urine. It combines thin-layer chromatographic separation and calorimetric estimation after direct diazo reaction of the developed spots scraped from the thin-layer chromatography plate.
Introduction The increased excretion of methylmalonic acid (MMA) in urine is a fairly sensitive index of vitamin B-12 deficiency [l-5] . Thin-layer chromatography (TLC), commonly employed for the detection of MMA in urine, provides only semiquantitative results. This brief communication describes a method for the quantitative estimation of urinary MMA by thin-layer chromatography, followed by diazo reaction of Giorgio and Plaut [ 61. Methods A 24-h specimen of urine was stored at 4°C with toluene as a preservative. An aliquot of 20 ml was acidified to pH 1.0 by the addition of 10 N sulphuric acid. Two aliquots of 10 ml were extracted 5 times with diethyl ether. The pooled extracts were treated thrice with chloroform, and the final extract was evaporated to dryness. The moisture-free residue was dissolved in 4 ml of chloroform. As the MMA content of normal urine is very small, it is necessary
* Corrcspondcnce of
Pathology,
should
be
Postgraduate
addressed Instituk
to:
Dr. of
K.C. Mrdical
Das,
Head
Education
Division and
of Hacmatology, Research.
Department
Chandigarh,
India.
to extract a 200 ml aliquot of a 24-h specimen of normal urine for quantitative estimation. 50 ~1 of this aliquot were applied (in duplicate) adjacent to the authentic standards of MMA on a dried and activated Silica gel G plate (TLC plate). This was developed in a bu.ffer tank containing the solvent system described by Dreyfus and Dube [7]. The plates were allowed to dry completely, and then sprayed uniformly with 2,7-dichlorofluorescein solution (0.1% in 95% ethanol). The spots of MMA were visualised under ultraviolet light and their outlines were mapped. The marked spots were scraped into individual test tubes. Equivalent areas of silica gel from the plates were scraped (in duplicate) into test tubes as blanks. The scraped spots collected in the test tubes were directly diazotised by the method of Giorgio and Plaut [6]. Attempts to elute MMA from the scraped silica gel spots using various solvents were unsuccessful. MMA appeared to adhere to the silica gel very tenaciously. Direct diazo reaction with scraped silica gel containing MMA produced an emerald green color in the supernatant. This was decanted after centrifugation. The absorbance of the supernatant was measured at a wave length of 620 nm. Results and comments It was possible to detect 10 ,ug of MMA on a TLC plate using the dichlorofluorescein spray and visualisation under ultraviolet light. The diazo reaction of the scraped spots yielded an emerald green color in the supernatant proportional to the concentration of MMA. The standard curves drawn by plotting the absorbances against different concentrations of the standard MMA are shown in Fig. 1. The slope of the standard curve, obtained after subjecting MMA to TLC (Fig. la) was slightly more inclined to the abscissa as compared to that obtained with similar concentrations of the standard subjected to direct diazo reaction without chromatography (Fig. lb). However, although TLC appeared to modify the slope of the curve, it was still linear, and could be utilised for the quantitative estimation of MMA in unknown samples of urine. In 5 experiments, 90 to 95 percent of MMA added to normal urine was recovered by this method. The MMA content of the urine of 10 normal subjects estimated by the
04
0
IO METHYL
Fig. diazo
1. Standard reaction
20 MALONIC cuwes without
30
40
50
ACID IN MICROGRAM of methylmalonic TLC.
--+ acid.
a, DiaTo
reaction
of scraped
spots
from
TLC
plate;
b, Direct
265
procedure described above, ranged from 0 to 6 mg/24 h with a mean of 2.8 mg/24 h. In 10 patients with nutritional megaloblastic anemia associated with vitamin B-12 deficiency, the urinary excretion of MMA varied from 15 to 320 mg/24 h with a mean of 212 mg/24 h. The procedure is relatively simple, reproducible, and can detect small quantities of MMA present in samples of normal urine. References 1
Cox,
2
Brozotic,
E.V.
and
White.
M..
Hoffbrand,
A.M.
(1962) A.V.,
Lancet
ii, 853-856
Dimitriadov,
A.
and
Molin,
D.L.
(1967)
Br.
J. Haematol..
13
1021-
1032 3
Gompertz,
4
Barma,
D., L.A.,
Jones, Young,
Hywel, D..
J. and
Mcllmari,
Knowles. W.J.,
J.P.
Kahn,
(1967)
S.B.
Clin.
Chin
and Williams,
Acta,
W.J.
18,
(1963)
197-204 New
Engl.
J. Med.
144-146 5
Williams,
6
Giorgio,
A.J.
D.L..
7
Dreyfus.
P.M.
Spray, and and
G.A.,
Plaut, Dube.
Newman.
G.W.E. V.E.
(1965) (1967)
G.E.
and
J. Lab. Clin.
O’Brien. Clin.
Chim.
T.R.P.
Med.,
Acta.
15.
66,
(1969) 667476
525-528
Br. J. Nutr.,
23,
343-352
265,