High-resolution electrophoresis of urinary glycosaminoglycans: An improved screening test for the mucopolysaccharidoses

I*

ANALYncAL~Y

a-L

m

no-127

(1982)

mw’ groppof-storabe

are

drsordnscausedbyadctkbcyinaoear tbcaahcroftllealxymcsamc8medwitbtbe caof v (Gags). IlltlJepast,tbcMPs6ba~ban-ixuIlargclyontbebaaisofdinical~ YariousQattunsof~ typtsd glycanuria(I~)~wlKmoneoftlleIuIPssis ap6siiexplanationoft.hcprciscnting~ icalphcnotypc,tbe~tcstistotbepatialtfor~&The acid albumin tarbidity tat (3), Yarious dye spot tests (45). aml the -Ym cblodc(CPC)~tcst(6)a1equick screuhgtestsf~the~ ofcxcess GagiIltbcluiIlGbowmr,thtJ+tcstsare hlowntogivebothfakpaeitivcandfake negatk results (6,7)- hdicati~~, qualitathe pattansof~nriamaybe

pmsidqdbyrehthdysimple~or~ dmensmdcllmma~andpllomtk tecbnigpcs @-IO), but in our expaiencetbeMidircdiooalsclIarationsdo notclearlyrcsolYcalltberckYantGagspeckTvw&muhddcdrophorccasen&kSbdter-Witbbigb-~, butbasamajorreshi&mofoncsamplepcr tkdrophoreticmnamIistbcreforcpnspit-

abkasastxemkgpoadprc, Iopractiqthcrcarefour-patterns of glye?lria-dermatan sldfate (IBX hcpran sulfate (I-IS), or Ladan sulfate (KS) alone or DS+HS in combina-ofdrrhichmaybcassociated witbaspadicdckkncyinone(ormore) ofthe10ly!5ondalzymcs P==mtobehvohwdintbecatabolismofDS.HS, and KS (1,2). Tests that arc -tb==f andtbatdiablyidcatcadhrderinGag catabolismaodakoprovideaguidctotbe mostprobabkeIuymckaionarccownpkx andtlKd~notslIitabkforscmminglargc patientnumbcrs.Cappdkttiezd.(Il)ba~ rccedydescriiiancwdcctrop8ordn: mctbodforthescpara~ofarnnediRtis=Gqs+MW),HS,KS,cbondroi-

IMPROVED

SCREENING

TEST

FOR

MUCOPOLYSACCHARIDOSES

121

and the centrifuge tube and pellet drained by inversion for 30 min. The pellet, dissolved in 150 ~1 of 2 mol/liter LiCl, was mixed with 800 ~1 ethanol and transferred to a 3-ml conical centrifuge tube, held for 5 min at 25°C and centrifuged again at 15OOg for 10 min. The supernatant was aspirated and the precipitated Gag dried under a stream of dry nitrogen at 25°C. Electrophoresis of isolated urinary Gags. Gag pellets (approximately 5-20 pg uranic MATERIALS AND METHODS acid) dissolved in 20 ~1 water containing 0.5 Urine collection. Random samples of urine g/liter phenol red were loaded by two apobtained from MPSs patients (Hurler, plications at the origin (Fig. 1A) with a Scheie, mucopolysaccharidosis type I (MPS Multi-applicator (Shandon Scientific Co., I); Hunter, MPS II; Sanfilippo A and B, London, England) to a cut piece (60 X 94 MPS IIIA and MPS IIIB; Morquio A, MPS mm) of Titan III Zip Zone cellulose acetate IVA; Maroteaux-Lamy, MPS VI) and nor- plate (Helena Laboratories, Beaumont, mal controls were frozen without added pre- Tex.), previously soaked in 0.1 mol/liter barservative. All diagnoses of the MPSs were ium acetate buffer, pH 5.0, and lightly blotbased on extensive clinical and X-ray ex- ted with Whatman No. 1 paper to remove aminations and demonstration of the defi- excess buffer. The cellulose-acetate strip ciency of specific lysosomal exohydrolase was then placed on the plastic film-insulated activity in cultured skin fibroblasts and/or cooling plate (maintained at lS°C) of an peripheral blood leukocytes. electrophoresis tank (Model U77, Shandon Gag standards. H, C4S, C6S, and DS Scientific Co., London, England), containing (grade II, types A, C, and B, respectively) 1.O molfliter barium acetate (pH 5.0). Wicks and CPC were purchased from Sigma cut from Whatman 3MM paper were conChemical Company (St. Louis, MO.). HS ditioned by electrophoresis in 1.0 mol/liter from bovine lung and KS from human costal barium acetate buffer for 30 min, attached cartilage were gifts from Dr. A. Cifonelli to the cellulose-acetate strip, covered with (Department of Pediatrics, University of a layer of Parafilm (American Can Co., Chicago). Hyaluronic acid (HA) was pur- Greenwich, Conn.) and a sheet of foam plaschased from Miles Laboratories (Elkhart, tic, and an even pressure was applied with Ind.). KS was also isolated from bovine in- a stack of glass plates (weight, 950 g), as tervertebral disc (12). Barium acetate was shown diagramatically in Fig. 1B. The strip purchased from Ajax Chemical Company was subjected to electrophoresis for 5 min (Sydney, Australia) and Alcian blue 8 GX at a constant voltage of 200 V, removed from from Gurr, Searle Diagnostic (Bucks, Eng- the electrophoresis tank, immersed in 0.1 land). mol/liter barium acetate buffer (pH 5.0) Precipitation of Gags from urine. Urine containing 15% v/v ethanol for 2 min, blotwas centrifuged at 15OOg for 10 min at ted as before, replaced in the tank, and re25°C. The supernatant, equivalent to 10 electrophoresed at a constant voltage of 200 pmol creatinine, was mixed with an equal V for 30 min using the same wicks and tank volume of 0.2 mol/liter sodium citrate buffer, buffer as before. The cellulose acetate suppH 4.8, containing 1 g/liter CPC and in- port was once more removed from the eleccubated at 37°C for 30 min, centrifuged at trophoresis tank and this time immersed in 15OOg for 10 min, the supernatant decanted, 0.1 mol/liter barium acetate buffer (pH 5.0)

tin 4-sulfate (C4S), chondroitin 6-sulfate (C6S), DS-in a monodimensional run that makes use of their different solubilities in ethanol and electrophoretic mobilities in barium acetate buffer. We have used this concept to develop a simple, convenient, and reproducible procedure for screening urine from patients under assessment for one of the many different types of MPSs.

122

HOPWOOD

AND HARRISON

FIG. 1. (A) Origin and wick position dimensions on the cellulose acetate strip used for electrophoresis. (B) Arrangements used for cooling of the cellulose acetate strip during electrophoresis. Dimensions are expressed in millimeters.

containing 50% v/v ethanol for 2 min and blotted. The strip was reelectrophoresed as before by applying a constant voltage of 200 V for 10 min. The strip was then stained in a 0.25% w/v aqueous Alcian blue solution for 15 min, destained in 0.18 mol/liter acetic acid, finally washed in deionized water, and dried at 25°C. Each channel was scanned with transmission densitometry at 560 nm with a slitwidth of 0.5 mm and a scan speed of 2 in./min in a Schoeffel Model SD 3000 spectrodensitometer and SDR 303 recorder (Schoeffel Instrument Corp., Westwood, N. J.) fitted with a Series 200 Disc integrator (Disc Instruments, Inc., Costa Mesa, Calif.). Uranic acid was determined by the carbazole method of Bitter and Muir ( 13) using D-glucurono-y-lactone as standard. Creatinine was determined on a CentrifiChem 400 centrifugal analyzer (Union Carbide Corp., Tuxedo, N. Y.) by the method of Fabing and Ertingshausen ( 14). RESULTS

Isolation of Gags from urine. A simhr maximum yield of uranic acid was obtained from urine from a Sanfilippo I? patient and

an age-matched normal control using CPC concentrations of 0.5 to 1.0 g/liter (Fig. 2). However, the relative levels of HS in the total Gag precipitates were different and appeared to vary from 22 to 28% and from 50 to 66%, respectively, over the range of CPC concentrations evaluated (Fig. 2). To evaluate any loss of Gag that may result from variations in time and temperature during the CPC precipitation step, urine sampled from a Sanfilippo patient and a normal individual were processed using changes in the standard procedure shown in Table 1. The relative proportion of HS or the yield of Gag precipitate did not change (Table 1). Identification of Gags excreted by MPSs patients. The present method clearly separated a mixture of the standard Gags H, DS, HS, C4S, C6S, and KS into individual components (Fig. 3). Standard DS contained two components, DSl and DS2, which represent 60 and 40%, respectively, of the total DS. The difference in composition between DSl and DS2 is not known. The only other Gag known to migrate in the DS2 zone is HA. However, HA was shown to consistently migrate 1 to 2 mm slower than DS2, to stain

r

1

-60

iz -60I -.OB t

FIG. 2. Gag composition and yield (rg uranic acid/ IO pm01 creatinii} as a function of CPC concentration. Yield of Gag from urine collected from a normal control (A) ati a .%n&p~c B patient (0) are shown tog&k with the proportion of HS (A and 0). respectively.

IMPROVED TABLE

SCREENING

TEST FOR MUCOPOLYSACCHARIDOSES

I

MPS I, II, and VI urines, The DS2 content of MPS VI urine was generally higher than that observed in MPS I and II urine. MPS I and II urine, compared to MPS VI, contained higher proportions of HS. The overall Gag patterns in MPS I and II urine were similar. The electrophoretic pattern obtained for the Gag precipitated from MPS IIIA and IIIB urine was clearly different from both normal and the other MPSs urines (Figs. 3 and 4). The relative proportion of HS to the total Gag in MPS III urine was greater than that found in normal and other MPSs urine (Table 2). Heparin was present in three out of the five MPS IIIA urines and was not seen in any MPS IIIB urine. The HS component in MPS IIIA urines exhibited a slightly slower electrophoretic mobility compared to the HS in MPS IIIB urine. KS was clearly separated from other Gags and, as expected, MPS IVA urine contained elevated proportions of both KS and C6S as compared to normal values. Although most urine patterns shown in Fig. 3 are clearly separated, the densitometer scan (Fig. 4) generally failed to resolve C4S and C6S bands from each other. In fact, there are often three to four different bands clearly seen in the CS region

EFFECT OF CPC INCUBATION TEMPERAIWRE AND TIME ON THE MEASURED PROPORTION OF HS IN THE COMPOSITION OF PRECIPITATED GAGS FROM NORMAL URINE

Temperature v-3 4 4 25 25 31 37

Heparan sulfate (%) (duplicate assay values)

Time (h) I 20 1 20 1 20

20, 23, 27, 23, 22, 20,

23 21 21 23 26 22

with Alcian blue to a lighter color, and unlike DS2, not to stain in Alcian blue containing 0.2 mol/liter MgC&. Figure 3 shows the pattern of Gags in urine from two normal individuals and each of the commonly observed MPSs. The major Gags detected in urine from normal subjects were C4S, C6S, and HS. KS and DS were generally not detected in urine from normal controls. Gag patterns from the urine of 47 MPSs patients were readily distinguished from normal individuals (Table 2 and Fig. 4). Two DS bands (DSl and DS2) were observed in

+ KS

--c

CtS cd5 QSZ

-c + -

HS

-I

r1

=

N

I

123

II

IIIA

Ill6

IVA

VI

N

FIG. 3. Electrophoretic separation of Gags isolated from urine provided by two normal individuals and MPSs type I, II, IIIA, IIIB, IVA, and VI patients. The mobility of standard Gags, H, DS (DSI and DSZ), HS, C4S, C6S, and KS are arrowed.

124

HOPWOOD

AND HARRISON TABLE

COMPOSITION MPSs

type

I (n = 10) II (n = 11) IIIA (n = 7) IIIB (n = 6) IVA (n = 6) VI (n = 7) (II/IVA)-Mixture (d = 29)

2

OF GAGS PRECIPITATED

FROM URINE

BY CPC

H

DSl

HS

DS2

cs

KS

nd-5 nd nd-13 nd nd nd

11-42 21-32 nd-4 nd-4 nd-4 1l-29

lo-42 29-46 50-78 45-82 6-19 nd-7

22-33 14-32 nd nd nd-12 27-57

9-23 12-24 24-50 18-55 61-81 14-54

nd nd nd nd 9-20 nd

nd

14-21

18-24

IO-18

34-45

9-13

nd nd nd nd t5

nd nd nd nd <5

22 23 42 31 5-39

nd nd nd nd nd

73 77 68 69 61-95

5 nd nd nd nd

Other lysosomal storage disorders GM,-gangliosidosis GM,-gangliosidosis cw-Mannosidosis Mucolipidosis type II Normal (n = 73)

Note. Proportion of each Gag was determined from the densitometer scan of the electrophoretic patterns. nd, ~1% detected, n, number of individuals; d, number of assays on an arbitrary mixture of urine from an MPS type II and an MPS type IVA.

of some urine patterns shown in Fig. 3. A normal Gag pattern was obtained for urine from patients with clinical phenotypes that are often confused with the MPSs but result from deficiencies of other lysosomal enzymes (Table 2). DISCUSSION

The method described here is an extension and simplification of that previously developed by Cappelletti et al. (11) and is applied as a screening test for the detection of the MPSs. It enables clear resolution of the CPC-precipitable DS, HS, CS, and KS species based on their different sulfate content, structure, and solubility in ethanol. Because the method can be applied to random samples of urine, it is especially useful in infancy, when 24-h collections are most difficult to obtain. In normal urine and in MPSs urine, approximately 20-40%, 50-70%, respectively, of the total urinary Gags have molecular

weights that are high (>3000) enough to precipitate with CPC (15). It is therefore emphasized that this method gives only the distribution of those urinary Gags with molecular weights greater than approximately 3000. The test is of value in the diagnosis of the MPSs, particularly in cases where the more conventional tests are falsely negative or positive. With such tests, there is a high false negative diagnostic rate for MPSs patients that have been subsequently diagnosed by enzyme assay (15). This is particularly so for the Sanfilippo syndromes, where out of our last 11 enzymatically proven cases more than half had negative glycosaminoglycanuria when assessed by CPC turbidity (6), acid albumin (3), and Azure A spot tests (5). Similarly, two out of five enzymatically proven MPS IVA and two out of six proven MPS VI patients were falsely negative according to these tests for an MPSs diagnosis. These patients are normally not detected and represent false negatives in tests that measure only the quantity of Gag excreted.

IMPROVED

SCREENING

TEST FOR MUCOPOLYSACCHARIDOSES

ab DISTANCE

cd FROM

e

fg ORIGIN

(mm)

FIG. 4. Densitometric tracings of electrophoretogram separations of Gag isolated from the urine of a normal individual (panel C) and MPSs type I, II, IIIA, IIIB, IVA, and VI patients (panels D, G, E, H, F, and I, respectively). The mobility of each standard Gag is arrowed a, b, c, d, e, f, and g for heparin, DSl, HS, DS2, C4S, C6S, and KS, respectively. Tracings from a separation of a standard mixture of purchased DS, HS, C4S, C6S, and KS, and an arbitrary mixture of urine from MPS type II and type IVA patients are shown in panels A and B, respectively.

125

126

HOPWOOD

AND HARRISON

When the present method was used to assess these same urine samples, however, all were clearly positive and provided a clear and accurate guide to the enzymological investigation. Breen et al. ( 16) quantitated Alcian bluestained Gags in various zones on cellulose acetate using a scanning densitometer. Recently, several methods have been described in which the amount of dye-Gag complex is measured following elution from the cellulose-acetate strip with solvent ( 17- 19). Compared to scanning by densitometer, these procedures are reported to increase the accuracy and reduce the variability of quantitative data. We chose to use scanning densitometry because of its relative simplicity and our need to process a large number of samples. Separate aliquots from an arbitrary mixture of urine from MPS II and IVA patients were processed through the standard procedure a total of 29 times as controls for other urines under test over a period of 3 months. The range of values shown in Table 2 for this control urine indicates that the variation introduced by the method of isolation, electrophoresis, and quantitation of Gags in urine does not invalidate the procedure as a diagnostic guide. The Alcian blue-binding capacities of purified preparations of CS, KS, HS, and DS have been shown to be linearly related to Gag concentration (17-19; J. R. Harrison and J. J. Hopwood, unpublished work). The upper limit for the accurate determination was reached when the capacity of the cellulose acetate membrane to retain the dyeGag complex was exceeded. This was greater than approximately 3 pg of C4S in the system used to generate the data in Table 2 (Harrison and Hopwood, unpublished work). The amount of Alcian blue bound to Gag is related to the number of negative charges, that is, sulfate and carboxyl groups, per repeating disaccharide unit. CS, DS, and HS would be expected to bind approximately 2, KS approximately 1, and H approximately

3-4 Alcian blue equivalents per repeating disaccharide unit. An allowance for the different extent of dye binding has not been made in calculation of the data shown in Table 2. Thus, KS content relative to the total Gag is probably higher and that of H probably lower than indicated in Table 2. Several urine samples contain an extra Alcian blue positive component with an electrophoretic mobility either between DS 1 and HS (11 mm from the origin) or similar to DS2. In two cases, repeat urine samples collected after the cessation of drug therapy indicated normal Gag patterns. Pennock et al. (7) observed temporarily raised CPC turbidity values in urine from children on antibiotics or anticonvulsant therapy. These materials may result from the sulfation of drug metabolites and should not be confused with the connective tissue Gags under study if a control urine is processed along side the urine samples under test. As a control, we routinely use an arbitrary mixture of urine from MPS II and IVA patients to indicate the relative efficiency of the CPC precipitation step and the mobility of all major Gag species (Table 2 and Fig. 4B). We recommend the described method as a simple, reproducible micromethod suitable for routine laboratory separation, identification, and quantitation of urinary Gags to provide a guide to the nature of the enzyme deficient in the MPSs. ACKNOWLEDGMENTS This work was supported in part by grants from the Research Trust of the Adelaide Children’s Hospital and The Channel 10 Children’s Medical Research Foundation of South Australia, Inc.

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TEST FOR MUCOPOLYSACCHARIDOSES

3. Carter, C. H., Wan, A. T., and Carpenter, D. G. (1968) J. Ped. 73,217-221. 4. Berry, H. K., and Spinanger, J. J. (1960) J. Lab. & Clin. Med. 55, 136-142. 5. Pennock, C. A., Mott, M. G., and Batstone, G. F. (1970) Clin. Chim. Acia 27, 93-97. 6. Pennock, C. A. (1969) J. Clin. Pathol. 22, 379380. 7. Pennock, C. A., White, F., Murphy, D., Charles, R. G., and Kerr, H. (1973) Acta Paediat. Sand. 62,481-491. 8. Humbel, R., and Chamoles, N. A. (1972) Clin. Chim.

Acta 40, 290-293.

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19. Gold, E. (1979) Anal.

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