Clinica Chimica Acta, 203 (1991) 211-224 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50

211

CCA 05135

sigh-pcrformanc~ liquid chromatography of urinary oligosaccharides in the diagnosis of glycoprotein degradation disorders Frits A. Hommes and ~olykutti

Varghese

Department of Biochemistry and Molecular Biology, Medical Coiiege of Georgia, Augusta, GA (USA)

(Received 4 March 1991; revision received 31 August 1991; accepted 3 September 1991) Key words: HPLC, Urinary oligosaccharides; Glycoprotein degradation

Summary

Urinary oligosaccharides can be separated by high-performance anion-exchange chromatography using a Dionex CarboPac PA1 column, elution with aqueous sodium hydroxide and sodium acetate soiutions and detection by pulsed amperometry. Each of the urines of patients with glycoprotein degradation disorders yielded a pattern of oligosaccharide excretion unique for that disorder, facilitating an unambiguous diagnosis. The method is sensitive (10 ~1 of urine required) and fast (40 min).

Introduction

The analysis of oligosaccharides excreted in the urine was pioneered by Humbel [1,21using thin layer chromatography and further refined by Sewell [3,4]. Variations on these methods using different stains to detect fun~ional groups attached to the oligosaccharides have been used [51.The technique allows a rapid diagnosis of glycoprotein degradation disorders, such as j%mannosidosis (McKusick 248500), and a-mannosidosis (McKusick 2485101, fucosidosis (McKusick 2300001,aspartylglucosaminuria (McKusick 2084001,Schindler disease (McKusick 104170), sialidosis (McKusick 2565501, galactosialidosis (McKusick 2565&I), GM-1 gangliosidosis (McKusick 230500), Sandhoff disease (MCI&sick 2688~), as well as some other conditions associated with the excretion of compounds likewise giving rise to Correspondence to: Frits A. Hommes, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100, USA.

212

specific bands in the thin layer chromatogram, such as Salla’s disease (McKusick 268740) or Pompe’s disease (McKusick 232300; 6). Several attempts have been made at analysis of oligosaccharides by column chromatographic methods, including HPLC, with limited success due to incomplete separations and difficulties in detection. Combined use of pellicular anion exchange resins operating at high pH values for the separation of carbohydrates at high resolution and pulsed amperometric detection of carbohydrates overcomes these problems [7,8]. In this communication we describe the application of these principles to the analysis of urinary oligosaccharides. Materials

and methods

Chemicals

Monosaccharides and disaccharides were purchased from Sigma (St. Louis, MO, USA), oligosaccharides from BioCarb Chemicals, Lund, Sweden. Sodium acetate (HPLC grade) and sodium hydroxide (50%, w/w) were obtained from Fisher Scientific (Norcross, GA, USA). Samples

Random urine samples were collected from enzymatically proven cases of the genetic diseases under investigation. They were kept frozen at -20°C until analysis or were lyophilized for transport and reconstituted with water to their original volume. Separation and detection of oligosaccharides

Urine samples were filtered through Gelman Acrodisc 3 (0.45 pm) prior to chromatography. The carbohydrates were analyzed on a Dionex HPLC apparatus, model BIOO LC, equipped with a pulsed amperometric detector (E, = O.O5V, T, = 480 ms; E, = 0.6OV, T2 = 300 ms; E, = -0.6OV, T3 = 200 ms). Volumes of 10 ~1 were injected. The carbohydrates were separated on a CarboPac PA1 column (3 x 250 mm), equipped with a CarboPac PA guard column at a flow rate of 1.0 ml per min. The column was eluted for 15 min with 90 mmol/l NaOH containing 15 mmol/l sodium acetate, followed by a linear gradient during the next 15 min to 200 mmol/l NaOH containing 600 mmol/l sodium acetate. This high concentration of eluent was maintained for 10 min, after which the system was reequilibrated with the first eluent for 10 min. Thin layer chromatography of urinary oligosaccharides was carried out as described by Sewell [4]. Results

Normal urines contain at least 60 compounds which can be detected by the pulsed amperometric technique. Figure 1 gives an example of such a chro-

213

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~,1,,,/1,,,,

0.00

1

I,

I

/

I,

I

c

I

I,

1

,

f

30.00

20.00

10.00

!

/

I,

I

/

I

/

,

11

40.00

11

11

If

I 50.00

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Fig. 1. HPLC of urinary carbohydrates of a normal 6-mth-old baby. Experimental conditions were as described in ‘Materials and Methods’. The creatinine content of the urine was 1.06 mmol/l.

matogram. The relative proportions of these fractions vary widely between urines, even when normalized on the creatinine content of the urines (Table I>. High values for the standard deviations were found, also when the area was expressed TABLE I Retention times and areas of the major fractions detectable described in ‘Materials and Methods’ Retention time (min)

SD (min)

1.35 1.54 2.14 2.37 2.77 3.26 3.84 4.88 8.44 21.45 24.66 27.85 29.08 31.72

0.00 0.03 0.04 0.02 0.01 0.02 0.06 0.05 0.08 0.10 0.03 0.11 0.05 0.03

Area (rel. units) 39,197

I 21,819 34,059 16,816 22,861 20,158 39,123 41,818 50,220 11,178 27,228 28,984 16,408 30,791

in urine by pulsed amperometty

as

&)I. units) 22,779 78,795 23,823 7,803 28,258 9,694 23,165 14,576 12,112 7,156 18,559 8,748 13,139 21,119

Mean and SD are given for the retention times and for the areas, normalized for the creatinine content, for 10 normal urines.

214 TABLE II Retention Methods’

times of some mono- and disaccharides

chromatographed

Compound

Retention time (min)

Sorbitol Trehalose Fucose N-Acetylglucosamine N-Acetylgalactosamine Galactose Glucose Fructose Lactose Isomaltose Sorbose Sucrose Maltose Sialic acid Galacturonic acid Glucuronic acid

2.00 2.32 2.38 3.48 3.80 3.80 3.82 4.30 4.32 5.98 6.22 6.28 14.38 21.47 24.07 24.70

as described in ‘Materials and

Fig. 2. HPLC of the urine of a GM-1 gangliosidosis patient (creatinine: 1.8 mmol/l). Experimental conditions were as described under Materials and Methods. Fractions uniquely increased for this condition have been numbered: fraction 1, GM-1 ganglioside, RT = 6.70 min; fraction 2, RT - 33.37 min; fraction 4, RT = 34.52 min; fraction 5, RT = 36.38 min, fraction 6, RT = 38.68 min.

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on a volume basis. The reproducibility is, however, very high as illustrated by the low values for the standard deviation of the retention times. A precise determination of retention times of the fractions is therefore possible. There are a number of carbohydrate and carbohydrate derivatives, which may appear in urine. The retention times of some of these compounds are listed in Table II. These compounds elute from the column relatively early under the given conditions, with the exception of the acidic derivatives, such as glucuronic acid, and galacturonic acid. Sialic acid likewise elutes relatively late. The chondroitin sulfates (4 and 6), dermatan sulfate, keratan sulfate and heparan sulfate did not give a signal, neither did any of the amino acids, when chromatographed under the present conditions. Figure 2 shows the chromatogram of the urine of a patient with p-galactosidase deficiency (GM-1 gangliosidosis). There are at least 5 fractions which are uniquely

GM-l gangliosidosis Control Sucrose GM-l ganglioside Fig. 3. Comparison of the chromatogram of a GM-1 gangliosidosis patient, normal control, GM-l ganglioside and sucrose. The normal control had a creatinine content of 2.38 mmol/l. Sucrose and GM-l ganglioside concentrations were 1 mg/ml. Experimental conditions as in ‘Materials and Methods’.

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increased in this condition, with retention times of 21.56 + 0.16, 33.10 k 0.02, 34.22 + 0.06, 36.02 f 0.03, and 38.35 f 0.09 min (n = lo), respectively and which are barely detectable in normal urines. The reproducibility of the elution times of these fractions is the same as those given in Table I for the fractions observed in normal urines, as illustrated by the SD of the elution times. The GM-1 ganglioside elutes at 6.70 min (c.f. ref. [7]) and separates therefore from a fraction eluting at 6.28 which is observed in some urines and which is probably sucrose (c.f. Table 11). This is more clearly illustrated in Fig. 3 where the GM-l gangliosidosis urine is compared with a normal sucrose containing urine, sucrose and the GM-1 ganglio-

Aspartylglucosaminuria Aspartylglucosaminuria

1 2

Fig. 4. Comparison of the chromatograms of the urines of hvo aspartylglucosaminuria patients. The condition characteristic fractions are indikated by arrows with retention times of 6.70 and 23.27 min, respectively. Experimental conditions as described in ‘Materials and Methods’.

217

side. Although the GM-1 ganglioside can be clearly recognized, what makes the chromatogram characteristic for p-galactosidase deficiency are the fractions eluting later from the column and which are obviously different from the GM-l gangIioside. In this respect, the HPLC technique is not different from the thin layer chromatography technique. The GM-1 ganglioside has a Rf value of 0.92 relative to lactose but the band observed at that position with a GM-l gangliosidosis urine is not the most prominent [4,.5]. A similar high degree of reproducibility was observed with the urine of another GM-l gangiiosidosis patient. In this case the GM-l ganglioside was clearly sepa-

(A)

6 s

Sandhoff Control II Control I Fig. 5. NPLC of the urines of a Sandhoff disease patient (creatinine: 1.06 mmol/l) and two normal controls, creatinine 1.06 mmol/l for control 1 and 0.87 mmol/l for control II (Fig. 5A) and the composite of the chromatograms of the urines of the Sandhoff patient of Fig. 5A and an additional Sandhoff patient (Fig. 5B). The numbered fractions indicated in Fig. 5B refer to the same fractions as indicated in Fig. 5A and have the following retention times in min: 1: 7.48, 2: 9.78; 3: 10.67; 4: 19.82; 5: 21.&; 6: 24.47; 7: 26.38. ~perimental conditions were as described in ‘Materials and Methods’. Fractions uniquely increase&in these conditions have been indicated by an arrow.

Fig. S. fcontinued).

rated from other ear& efuting &om~ounds with an elution time of 6.65 f 0.18 min (rt = 10). The other 5 fractions eluting Iater yietded retention times of 21.75 f 0.11, 33.12 &,O&3,34.24 -I_O&5,36.03 $: 0.04 and 38.31 rt:0.08 min (n = 101,respectively. Simifar results were obtained witb the urines of two o&her GM-1 gangIiosidosis pati&&, ~-Ac~~ylgI~cosaminyI asparagine and N-ace&l-lactosaminyl asparagine have retention times of 6.70 and 9.37 min, respectively. Both caulpou~ds have been demous~ra~~ to be present in urine of aspa~yIglu~~aminuria patients @I. The former could clearfy be re#gnized in the ~p~~IgIucosamiuuria sample anafyzed, but the latter was present - if at BII - in such iow con~n~ratio~ that it escaped detection. However, aspar~lgiu~osaminuria did yield an unique fraction with a retention time of 23.27 min. Figure 4 shows a comparison of the urines of two aspa~IgIu~~inu~a patients. The &h~a~~eristic fractions overlap in this composite figure both q~an~i~a~~elyand quaIi~a~iveIy.The former is nut necessariIy the

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Sialidosis Control IV Control III Fig. 6. HPLC of the urines of a sialidosis patient (creatinine 1.06 mmol/l) and two normal controls (creatinine 1.06 and 1.68 mmol/l, respectively). The fractions uniquely increased in this condition are indicated by arrows and have retention times of 22.45, 23.80 and 24.27 min, respectively. Experimental conditions as described in ‘Materials and Methods’.

case in all patients in this and in the other conditions investigated, but the characteristic fractions do elute at the times specified. Each of the conditions examined gives rise to a specific pattern which allows an unambiguous identification. Figure 5 illustrates this for the urine of a patient with a deficiency of the P-subunit of hexosaminidase (Sandhoff disease, Fig. 5A) while Fig. 5B shows the comparison with the urine of another Sandhoff patient. Additional examples of oligosaccharide excretion patterns are given for sialidosis (Fig. 61, Schindler’s disease (c.f. ref. [lo], Fig. 7) and two fucosidosis patients (Fig. 8). Table III summarizes the retention times of fractions uniquely increased for the various conditions. The urine of the sialiluria patient showed one single abnormal fraction at 21.47 min, the same elution time as that of sialic acid (Table II). One abnormal fraction,

220

Fig. 7. HPLC of the urine of a Schindler’s disease patient (creatinine: 0.93 mmol/l). The fractions uniquely increased in this condition are indicated by arrows and have the following retention times in min: fraction 1: 6.00; fraction 2: 15.62; fraction 3: 21.87; fraction 4: 22.55; fraction 5: 23.52; fraction 6: 25.80; fraction 7: 37.37. Experimental conditions as described in ‘Materials and Methods’.

TABLE III Retention times of fractions uniquely increased conditions

in glycoprotein degradation

disorders and related

Condition

Retention time (min)

Aspartylglucosaminuria (2) a Fucosidosis (2) Galactosialidosis (2) GM-l gangliosidosis (3) cY-Mannosidosis (2) P-Mannosidosis (3) Mucolipidosis III (2) Multiple sulfatase deficiency (1) Pompe (1) Sandhoff (2) Schindler (1) Sialidosis (2) Sialiluria (1)

6.70; 23.27 2.63; 4.45; 6.30; 8.67; 24.42; 25.73; 28.90 8.63; 13.22; 21.00; 21.50; 21.68 6.70; 8.63; 13.25; 21.90; 33.37; 34.52; 36.38; 38.68 6.52; 24.50; 29.00; 31.60 4.40; 21.55; 22.10; 27.00; 31.48 6.55; 21.95; 23.15; 23.88; 29.72; 30.88 1.88; 22.65; 23.75; 26.00; 29.42; 30.12; 30.83 22.60 7.48; 9.78: 10.67; 19.82; 21.48; 26; 35 6.00; 15.62; 21.87; 22.55: 23.52; 28.80; 37.37 22.45; 23.80; 24.47 21.47

a The number of patients analyzed is given in parentheses.

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Fucosidosis-1 Fucosidosis-2 Fig. 8. Comparison of the chromatograms of the urines of two fucosidosis patients. The condition characteristic fractions are indicated by arrows and have the following retention times in min: fraction 1: 2.63; fraction 2: 4.45; fraction 3: 6.30; fraction 4: 8.67; fraction 5: 24.42; fraction 6: 25.73; fraction 7: 28.90. Experimental conditions as described in ‘Materials and Methods’.

a retention time of 22.60 min was observed in the urine of the Pompe’s disease patients. The tetramer of glucose which is observed in the urine of such patients [6] has the same retention time. The pattern of fractions detectable in urine by this method is rather constant when multiple urines of the same patient are analyzed. There are quantitative differences, but qualitatively the patterns are the same.

with

222

L

Urine 1

z

w

Urine 3

Fig. 9. Oligosaccharide pattern of three urines of a B-mannosidosis patient. The urines we collected over a period of 1 3/12 years. The fraction uniquely increased in this condition are indicated by arrows and have the following retention times (in mink fraction 1: 4.40; fraction 2: 21.55; fraction 3: 22.10; fraction 4: 27.00, fraction 5: 31.48. Experimental conditions as described in ‘Materials and Methods’.

It has been shown that newborns on breastfeeding excrete oligosaccharides in their urine as demonstrated by the thin layer chromatography technique [ill. That has been confirmed by the present method. It concerns particularly the fractions with retention times of 8.44, 24.66 and 31.72 min which are always observed in normal urine (c.f. Table I) but which are considerably more prominent in breastfed infants. In addition to these fractions, fractions eluting at 7.67, 9.17, 28.28 and 28.78 min, respectively, were observed, which are not always observed in normal urines, and if so, at very low levels. None of these fractions, however, are excreted in increased amounts in any of the pathological conditions examined (c.f. Table II). Similarly, urine of trauma patients contained increased levels of fractions with retention times of 21.58, 23.13 and 27.75 min, respectively, which are usually not

223

observed in normal urines, as well as fractions eluting at 24.66 and 31.72 min, which also occur in normal urine, but in considerably lower amounts. None of these fractions are observed in the pathological urines examined.

Discussion The diagnosis of disorders of the degradation of glycoproteins rests on the demonstration of a specific enzyme deficiency. To determine which enzyme should be tested for, a preliminary screening of the urine for oligosaccharides has been extensively used. Thin layer chromatographic procedures [l-61 offer a method of separation of the urinary oligosaccharides which in many cases yield a pattern of bands unique for a specific condition, especially when complemented with staining for glycopeptides [51. The interpretation of the pattern of bands is, however, not always easy and/or unambiguous. Identifi~tion of specific bands by a more quantitative parameter is provided by the retention times of the fractions obtained in HPLC over a CarboPac PA1 column. Each of the conditions tested did give rise to a set of fractions with unique retention times, which proved to be highly reproducible, facilitating an unambiguous identification of the condition. There are at least 60 fractions detectable in normaf urine with, the pulsed amperometric technique. It does not necessarily mean that these fractions contain (oligokaccharides. Any compound eluted from the column which can be reduced at the voltage applied will give rise to a signal and will be registered as a fraction. More fractions may be detectable at other voltages, depending on the redox potential of the compounds. It implies that this technique may detect compounds other than (oligo)saccharides, an area which is as yet unexplored. The concentration of many of the fractions detectable in urine are low, especially those eluting at higher sodium acetate concentrations. None of these fractions has been identified. This touches on the specificity of the method for the detection of disorders of glycoprotein degradation and related conditions. The demonstration of an abnormal pattern does not necessarily imply such a condition. An example is the increased excretion of the glucose tetramer in pancreatitis 1121. A confirming test by enzyme assay or DNA techniques should always be carried out. The individual fractions can easily be collected, hydrolyzed and the hydrolysate rechromatographed over the same column, using the same technique with slightly different gradient conditions to detect. monosaccharides. Alternatively the monosaccharides can be deri~tized for GC/MS (c.f. ref. 1131). In view of the high resolution of the HPLC technique for oligosaccharide detection in urine, a considerable improvement in the preliminary screening of patients with glycoproteinoses and related disorders has been accomplished. This high resolution will also facilitate the identification of other compounds detectable by the pulsed amperometric technique, including those of glycoprotein metabolism.

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Acknowledgements Thanks are due to Drs. K. Unui (Osaka, Japan), H. Taylor (Greenwood, S.C.), E. Zammarchi (Firenze, Italy), R. Gatti (Genoa, Italy), B. Gordon (London, Ont,, Canada), and J. Huymans (Rotterdam, The Netherlands) for urine specimens. References 1 Humbel

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