Panin, S. Nala, A. DaIl'Amico, L. Chiandetti, F. Zachello, C. Catassl,'. L Feud,1 and G. V. Coppa'. Ne describe a simple, rapid, precise, and sensitive spectro-.
2. Mancini quantitation nochemistry
G, Carbonara AD, Heremans JF. Irmnunochemical of antigens by single radial inununodiffusion. Imrnu1965;2:235-54.
1. Willoughby EW, Lambert A. A sensitive silver stain for proteins .n agarose gels. Anal Biochem 1983;130:353-8.
CLIN. CHEM.
32/11, 2073-2076
4. Peterson
PA, Evrin P-E, Berggard I. Differentiation of glomerular, tubular, and ordinary proteinuria: determinations of urinary excretion of beta2-microglobulin, albumin, and total protein. J Clin Invest 1969;48:1189-98. 5. Davey P0, Gosling P. Beta2-microglobulin instability in pathological urine. Clin Chem 1982;28:1330-3.
(1986)
Simple Spectrophotometric Quantification 6lycosaminoglycan Sulfates .
Panin, S. Nala, A. DaIl’Amico,
L. Chiandetti,
F. Zachello,
Ne describe a simple, rapid, precise, and sensitive spectro)hOtometric method for measuring urinary glycosaminoIycan (GAG) sulfate excretion. The GAG sulfates are preipitated with cetylpyridinium chloride, resuspended in water, nd mixed with the basic dye 1 ,9-dimethylmethylene blue to )roduce
3AGs.
a complex Absorbance
with the polyanionic molecule of sulfated is read at 535 nm. The standard curve for
eaction was linear up to 12 of the different GAGs: ermatan sulfate, heparan sulfate, keratan sulfate, chondroiin 4-sulfate,
and chondroitin
6-sulfate.
Within-
and between-
un precision (CV), measured at three different GAG concen:rations (normal and pathological), varied from 1.6% to 2.5% nd from 1.8% to 4.5%, respectively. Analytical recovery anged from 71% to 107%. Urinary GAG excretion, meaured by this procedure, correlates (r = 0.837; p ) .
id 2.1%,
respectively. Between-run precision, determined om data for 10 replicate analyses of six urine collections ntaining increasing amounts of GAGS (3.1 to 199.7 mg), nged from 1.8% to 4.5%. Analytical recovery. When various amounts of GAGS were ided to 24-h urine specimens from an healthy individual id from two affected children (one with mucopolysacchari)Sis H and one with mucopolysaccharidosis VI), between L% and 107% of the added GAGS were accounted for (Table Correlation with the borate-.carbazole method. Results by te DMB method (x) were linearly correlated (r = 0.837; p 0.001) with those by the borate-carbazole procedure of
Table 1. Analytical Recovery of C-4-S by the DMB Method mpIe
Sulfated
no.
and vol, mL
cs added,
mg
2.5
Expected
7.13
10.08
4.23
5.08 2.58
Recovery,
%
0. 2, 25
10 5
2.5 0.5 0.25 12.5
71 83
1.08
94 93
0.58 0.33
91 87
10.42 6.07
12.29
85
7.29
83
4.40 3.44
4.79
92
3.29
2.93 2.70
2.79 2.54
104 105 106
11.13
73
6.13
81
3.63
82 90 99
2.44 1.01 0.53 0.29 2.29
0.5 0.25
Table
2. Urinary with
0.5 0.25 1-3 contained
66.
2.13
GAG
excretion,
Affected mg/24 h
Borate-carbazole
0MB
50.5
12.3 19.5
1.63 1.47 1.38 107 55.0, and 49.0 mg of sulfatedGAG per 24-h 1.61
GAG ExcretIon In 5 PatIents Mucopolysaccharldosls IV Total
1.13
8.6 4.96 2.97 1.92
10 5
ne.
Measured
0.08
10 5
uSamples
GAG,
mg/sample
lo. 1, 12.5
0.3,
dye is one of the most sensitive thiazine compounds, marked shift when complexed with GAG than do other thiazine dyes (6). The instability of the dye reagent,-GAG complexes has been overcome by Farndale et al. (7), who used a formate buffer instead of the dibasic citrate/ phosphate buffer recommended previously (6). To determine urinary sulfated GAGS, we introduced a precipitation step, to minimize any interference from salts and negatively charged compounds (e.g., glycoproteins, nucleic acids). CPC effectively precipitates GAGS (8) but also tends to coprecipitate glycoproteins. This problem can be overcome by using CPC concentrations greater than that recommended by Di Ferrante (9, 10). Moreover, CPC also precipitates urinary keratan sulfate (8). Because the color yield of the dye-GAG complex with increasing amounts of pure suspensions of various sulfated polisaccharides is remarkably similar, we could use C-4-S as a standard, without introducing a significant bias in the estimation of total GAGS (Figure 3). The color is stable long enough to ensure good reproducibility for routine laboratory work. Because the linearity of the reaction covers the range of physiological GAG excretion, only pathological samples need be diluted. When compared with the commonly used borate-carbazole procedure, the DMB method shows a linear correlation of results in the reference-interval range and about twice the sensitivity, considering that about 34% (by wt) of the C-4-S molecule consists of uromc acid residues. Analytical recovery of C-4-S added to urine samples was low only when the amounts of GAG added were far beyond the normal range. It is not clear whether this was because the CPC concentration may have been insufficient to precipitate all the suspended GAG molecules, or whether unknown interfering substance(s) were present. The reference range of 24-h urinary excretion of sulfated GAGS measured with the present technique is threefold higher than that measured with the borate-carbazole method. This ratio is increased for the urmnes from patients giving a more
_6
12.7 12.4 12.4
Reference
range
1.1-11.8
CLINICAL CHEMISTRY,
82.6 55.6 63.5 50.8 3.0-30.3
Vol. 32, No. 11, 1986
2075
affected by mucopolysaccharidosis IV. Because these methods have the CPC precipitation step in common, the difference must be partly related to the chemical composition of keratan sulfate, which contains little or no uronic acid. The reliability of the DMB technique in supporting the clinical suspicion of Morquio’s syndrome clearly is an improvement over the borate-carbazole method, which is prone to give false-negative or doubtful results. We therefore conclude that our procedure is simple, sensitive, and reproducible and can be used as a screening test in the diagnosis of inborn ermrs of GAG metabolism. We thank Dr. R. Ricci, Dept. of Pediatrics, Sacro Cuore University, Rome, for her cooperation in providing urine samples from her patients; Dr. L. Gentili for his greatly appreciated help in revising the manuscript; and L. Capuzzo and M. Silvestri for typing the manuscript. References
1. McKusick VA, Neufeld EF. The mucopolysaccharide storage In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS. eds. The metabolic basis of inherited disease, 5th ed. New York: McGraw-Hill, 1983:751-77. diseases.
2076
CLINICAL CHEMISTRY, Vol. 32, No. 11, 1986
2. Bitter T, Muir HM. A modified Anal Biochem 1962;4:330-4.
uronic
acid-carbazole
reactior
3. Whiteman P. The quantitative determination of glycosaminogly cans in urine with Alcian Blue 8 GX. J Biochem 1973;131:351-7. 4. Gold EW. A simple spectrophotometric method for estimatin glycosaniinoglycan concentrations. Anal Biochem 1979;99:183-8. 5. Pennock CA. A review and selection of simple laboratory meth ods used for the study of glycosarninoglycan excretion and thi diagnosis of the mucopolysaccharidoses. J Clin Pathol 1976;29:111-
23. 6. Taylor
KB, Jeifree GM. A new basic metachromatic dye, 1:9 methylene blue. Histochem J 1969;1:199-204. 7. Farndale RW, Sayers CA, Barrett AJ. A direct spectrophotomet nc microassay for sulphated glycosaminoglycans in cartilage cal tures. Connect Tissue Res 1982;9:247-8. dimethyl
8. Di Ferrante N. The measurement rides. Anal Biochem 1967;21:98-106.
of urinary
mucopolysaccha
9. Goldberg JM, Cotlier E. Specific isolation and analysis of muco polysaccharides (glycosaminoglycans) from human urine. Cm Chin Acts 1972;41:19-27. 10. Pennock CA, White F, Murphy D, Charles RG, Kerr H. Exces glycosaniinoglycan excretion in infancy and childhood. Acts Pae diatr Scand 1973;62:481-91.
