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Proton nuclear magnetic resonance spectroscopic detection of sialic acid storage disease.

Sialic acid storage disease (SASD) is a rare autosomal recessive lysosomal storage disorder characterized by excessive urinary excretion of free sialic acid and an accumulation of free sialic acid in skin fibroblasts (1). Clinically, two major forms exist: an infantile type with severe progression leading to early death (2), and a milder form (Salla disease) with a protracted course (3). The basic defect in both forms is a malfunction of a sialic acid transporter in the lysosomal membrane (4).

Because the clinical characteristics of SASD are very similar to those of other lysosomal storage diseases, differential diagnosis relies on detection of increased urinary sialic acid excretion by thin-layer chromatography of urine (5) and quantification by colorimetric analysis (6) or ion-exchange HPLC (7). However, each method has disadvantages. Colorimetric assays suffer from interference by 2-deoxyglucose, whereas HPLC methods require lengthy sample preparation.

Proton nuclear magnetic resonance ([sup.1]H-NMR) spectroscopy is a powerful tool for the structural elucidation and characterization of body fluid metabolites in inherited metabolic disorders (8-11). The technique is rapid, requiring minimal sample treatment. Sialic acid (free and as a conjugate) has been extracted from the urine of individuals with SASD and sialidoses, respectively, and characterized by [sup.1]H-NMR spectroscopy (12-14); however, the technique has not been used for direct diagnosis from unextracted urine.

We carried out [sup.1]H-NMR analysis of urine from five patients with SASD (two with the infantile form, one with the intermediate form, and two patients with Salla disease) and compared the urinary sialic acid excretion determined by colorimetric analysis and [sup.1]H-NMR spectroscopy.

Urine samples from patients with SASD were obtained as part of a selective screening program for inborn errors of metabolism. Clinical and diagnostic data on three patients have been described (15). The fourth patient had Salla disease and was studied at the age of 2 years. The fifth patient had a severe infantile form with clinical characteristics similar to the youngest patient, who died at 6 months. Increased free sialic acid was detected by thin-layer chromatography and quantified by colorimetric assay with correction (6).

Deidentified urine samples from 20 patients from a similar screening program without a confirmed diagnosis for an inherited disorder of metabolism and from 2 patients with confirmed diagnoses of sialidosis were also obtained.

Urine samples (2 mL) lyophilized for convenience of transfer to the NMR laboratory were reconstituted in 0.5-1.0 mL of [sup.2][H.sub.2]O containing 2.5 mmol/L sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionate (BDH Chemicals Ltd.) to act as a field frequency lock (to stabilize the spectrometer field frequency) and a chemical shift reference signal, respectively. Aliquots (0.5 mL) were then analyzed at 500 or 600 MHz in either a Jeol GSX500 or a Bruker AM600 spectrometer, respectively, at 20[degrees]C, with a pulse angle of 45 degrees (5 [micro]S), an acquisition time of 4.19 ms, and pulse repetition time of 5 s. Typically, 128-1024 scans were collected; 32 were sufficient for diagnosis (<3 min). The longest analysis time (1024 scans; 86 min), which improves the signal-to-noise ratio by a factor of [square root of 8] compared with 128 scans, was used initially to detect minor spectral components. [sup.1]H-NMR spectra were also obtained from two commercially available sialic acid standards: a synthetic preparation and sialic acid extracted from sheep submaxillary glands (Sigma-Aldrich Co. Ltd.). Two-dimensional correlated spectroscopy (COSY) spectra were also obtained on some samples to further characterize the putative sialic acid resonances.

Sialic acid excretion was calculated by comparing the peak height of the N-acetyl resonance(s) with that of the creatinine methyl group. For comparison of the magnitude of the N-acetyl resonance to that from the carbon-3 protons of sialic acid, peak areas were used (see below).

A [sup.1]H-NMR spectrum of sialic acid extracted from sheep submaxillary glands is shown in Fig. 1A. The spectrum contains a large singlet at 2.05 ppm, a small singlet at 2.03 ppm, and a pair of second-order multiplets (ABX system) at 2.24 and 1.84 ppm.

The peaks at 2.03 and 2.05 ppm were assigned to the N-acetyl protons of the [alpha] and [beta] anomers of sialic acid, respectively. The ABX systems at 2.24 and 1.84 ppm were assigned to the pyranose ring protons of carbon-3 of the [beta] anomer. These assignments agree with those of Brown et al. (14). Two resonances, at 1.6 and 2.73 ppm, were assigned to the carbon-3 protons of the [alpha] anomers. The signals at 3.5-4.1 ppm were from protons at the remaining positions in the pyranose ring.

Spectra for urine from an 11-month-old infant, screened for a suspected metabolic disorder, and from a 2-month-old infant with the severe form of SASD are shown in Fig. 1, B and C, respectively. The characteristic N-acetyl signal at 2.05 ppm is seen in the spectrum for the infant with severe SASD (Fig. 1C) but not in the spectrum for the infant with a suspected metabolic disorder (Fig. 1B). The pseudo-quartets from the ABX system of the protons from carbon-3 of sialic acid, at 2.24 and 1.84 ppm, are also visible only in the spectrum for the infant with severe SASD (Fig. 1C). In spectra from four of the study participants, both of these carbon-3 resonances were clearly visible.

Also shown in Fig. 1C is 1,2-propanediol, which is used as a solvent for barbiturate drugs (16).

Shown in Table 1 are the sialic acid:creatinine ratios calculated from the colorimetric assay (creatinine estimated by the Jaffe reaction) and by [sup.1]H-NMR spectroscopy (creatinine also measured by NMR). The [sup.1]H-NMR results were calculated from the sum of the N-acetyl peaks at 2.05 ppm ([beta]-sialic acid) and 2.03 ppm ([alpha]-sialic acid). The small ABX signals from carbon-3 protons of the [alpha] anomer of sialic acid could be detected in only two of the individuals. Sialic acid resonances between 3.5 and 4.1 ppm were obscured by other resonances in this region. However, some of these signals could be detected as cross peaks in the two-dimensional COSY spectrum (not shown). The ratio of the [alpha] and [beta] anomers of sialate is also shown in Table 1. In the patients with sialidosis, much smaller signals at 2.03-2.05 ppm were observed, but no corresponding resonances at 1.84 or 2.24 ppm were present (not shown). In the patients referred for suspected metabolic disorders, small resonances corresponding to N-acetylated compounds could be detected infrequently at 2.03 and 2.05 ppm.


The results demonstrate that the five cases of SASD studied gave [sup.1]H-NMR urine spectra that are diagnostic. The most prominent feature of the spectra is the signal at 2.05 ppm, although conclusions based solely on the finding of an abnormally high N-acetyl proton signal may be insufficient because several N-acetylated compounds may also be candidates. However, the presence of the characteristic carbon-3 proton resonances at 1.84 and 2.24 ppm should be decisive for free sialic acid detection because there are no other obvious candidates with this molecular configuration.

The NMR method can also distinguish between SASDs and the mucopolysaccharidoses, which lead to the excretion of oligosaccharides containing sialic acid residues (sialidoses). In these disorders, the sialic acid occurs as the terminal monosaccharide unit linked through carbon-2 to its neighbor in either a (2[right arrow]3) or (2[right arrow]6) linkage in various oligosaccharides, typically consisting of 9-11 units. The altered configuration at carbon-2 produces changes in the resonance positions of the protons on the adjacent carbon-3, to 1.80 and 2.76 ppm for the (233) linkage or 1.72 and 2.67 ppm for the (236) linkage (12) in these oligosaccharides.

The spectra from the two patients with sialidosis showed several much smaller signals in the 2.01-2.06 ppm region compared with those from the patients with SASDs, which are attributable not only to N-acetylneuraminate but also N-acetylglucose present in the oligosaccharides.

Two-dimensional COSY experiments on samples from both patients showed cross peaks between the 1.7-1.8 and 2.6-2.8 ppm regions as expected (not shown). There was good quantitative agreement between the colorimetric and NMR assays for four of the five samples. For the fifth sample, we have concluded that the NMR result is the more correct one for the following reasons: (a) the peak assigned to the [beta] resonance (the largest component) is at the appropriate frequency; (b) the sum of peak areas of the carbon-3 proton resources at 1.84 and 2.24 ppm is in the appropriate ratio to that of the [beta] peak (~1:3); (c) the ratio of [beta]/[alpha] anomers is 6.52, which is well within the range of values for this ratio in the other samples (Table 1); and (d) the colorimetric assay for this sample demonstrated considerable interference, so it is possible that some sialate was not detected. In some circumstances (not in the present case), it is possible that NMR may overestimate the sialic acid concentration if there are other N-acetylated metabolites present whose resonances overlap with that of sialic acid. Accurate quantification from the carbon-3 protons as an alternative is feasible only for the [beta] anomer; peak-area integrals are necessary because the line widths of the second-order ABX system differ from those of first order.

Although SASD is rare, the NMR method used is identical to that previously used for detecting other metabolic disorders and includes the concurrent determination of creatinine (17). This versatility is emphasized by the simultaneous observation of a dicarboxylic aciduria in one of the patients with Salla disease.

We are grateful to the University of London Intercollegiate Research Service (ULIRS) at Birkbeck, King's and Queen Mary and Westfield Colleges for NMR facilities. During part of this study, H.C.M. was supported by the UK Medical Research Council.


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(2.) Stevenson RE, Lubinsky M, Taylor HA, Wenger DA, Schroer RJ, Olmstead PM. Sialic acid storage disease with sialuria: clinical and biochemical features in the severe infantile type. Pediatrics 1983;72:441-9.

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(4.) Tietze F, Seppala R, Renlund M, Hopwood JJ, Harper GS, Thomas GH, et al. Defective lysosomal egress of free sialic acid (N-acetylneuraminic acid) in fibroblasts of patients with infantile free sialic acid storage disease. J Biol Chem 1989;264:15316-22.

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(7.) Mononen I. Detection of sialuria by cation-exchange high-performance liquid chromatography. J Chromatogr B Biomed Appl 1986;381:219-24.

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(10.) Burns SP, Woolf DA, Leonard JV, Iles RA. Investigation of urea cycle disorders by 1H NMR spectroscopy. Clin Chim Acta 1992;209:47-60.

(11.) Wevers RA, Engelke U, Heerschap A. [sup.1]H-NMR spectroscopy of blood plasma for metabolic studies. Clin Chem 1994;40:1245-50.

(12.) Dorland L, Havercamp J, Vliegenthart JFG, Strecker G, Michalski J-C, Fournet B, et al. 360-MHz 1H nuclear-magnetic-resonance spectroscopy of sialyloligosaccharides from patients with sialidosis (mucolipidosis I and II). Eur J Biochem 1978;87:323-9.

(13.) Haverkamp J, Van Halbeek H, Dorland L, Vliegenthart JFG, Pfeil R, Schauer R. High-resolution [sup.1]H-NMR spectroscopy of free and glycosidically linked O-acetylated sialic acids. Eur J Biochem 1982;122:305-11.

(14.) Brown EB, Brey WS, Weltner W. Cell-surface carbohydrates and their interactions. 1. NMR of N-acetylneuraminic acid. Biochim Biophys Acta 1975;399:124-30.

(15.) Sewell AC, Poets CF, Degen I, Stoss HA, Pontz BF. The spectrum of free neuraminic acid storage disease in childhood: clinical, morphological and biochemical observations in three non-Finnish patients. Am J Med Genet 1996;63:203-8.

(16.) Cady EB, Lorek A, Prentice J, Reynolds EOR, Iles RA, Burns SP, et al. Detection of propan-1,2-diol in neonatal brain by in vivo proton magnetic resonance spectroscopy. Magn Res Med 1994;32:764-7.

(17.) Iles RA. NMR analysis of clinical biofluids. In: Townshend A, ed. Encyclopaedia of analytical science. London: Academic Press Ltd., 1995:3532-44.

Adrian C. Sewell, [1] * Helena C. Murphy, [2,3,[dagger]] and Richard A. Iles [2,3][[dagger]]

[1] Department of Paediatrics, University of Frankfurt, 60596 Frankfurt am Main, Germany;

[2] Medical Unit and

[3] Department of Diabetes and Metabolic Medicine, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Whitechapel, London E1 1BB, United Kingdom;

* address correspondence to this author at: Department of Paediatrics, University Children's Hospital Frankfurt, TheodorStern-Kai 7, 60596 Frankfurt am Main; Germany; fax 49-069-6301-5229, e-mail;

[[dagger]] current address: Unit of Cell Regulation, Department of Diabetes and Metabolic Medicine, St Bartholomew's and the Royal London School of Medicine and Dentistry, Whitechapel, London, E1 1BB, United Kingdom
Table 1. Measurement of sialate (N-acetylneuraminate) in urine from
patients with SASD.

 N-Acetylneuraminate concentration
 (sum of [alpha] + [beta] anomers),
 mol/mol creatinine

Diagnosis Age Colorimetric [sup.1]H-NMR
 analysis analysis

Salla disease 16 years 0.12 0.14
Salla disease 24 months 0.43 0.42
Salla disease 11 months 0.69 0.56
ISASD (a) 6 months 1.39 1.28
ISASD 2 months 0.80 2.19
Mean ratio

Patients with <0.01-0.07

IV-S (Sigma)

Diagnosis [beta]/[alpha]

Salla disease 5.7
Salla disease 7.6
Salla disease 7.1
ISASD (a) 6.0
Mean ratio 6.6 [+ or -] 0.6
Patients with
 type 6.2
IV-S (Sigma)

(a) ISASD, infantile SASD.
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Title Annotation:Technical Briefs
Author:Sewell, Adrian C.; Murphy, Helena C.; Iles, Richard A.
Publication:Clinical Chemistry
Date:Feb 1, 2002
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