CLIN. CHEM. 35/3, 374-379 (1989)

374 CLINICALCHEMISTRY, Vol. 35, No. 3, 1989

DiagnosticTest for Mucopolysaccharidosis.I. DirectMethod for QuantifyingExcessive UrinaryGlycosaminoglycanExcretionChester B. Whltley, MIchelle D. Rldnour,KarlA. Draper, Charyl M. Dutton,and Joseph P. NeglIa

This direct method for quantifying excessive urinary glycos-aminoglycan excretion exploits the specific binding of 1,9-dimethylmethylene blue (DMB). The procedure obviatescumbersome and labor-intensive procedures for separatingglycosaminoglycansfromotherconstituentsof urine.Pediat-ric pharmaceutical formulations (except hepann), in concen-trations expected in urine, do not interfere with spectropho-tometry, nor does protein. Results can be expressed in termsof urinary creatinine; thus the test is applicable to very smallurine specimens (0.1 mL), such as those obtainable fromneonates.In a pilot study, results of the direct DMB test for 48urine specimens agreed with the clinical diagnosis, andquantitative measurements correlated moderately (r = 0.76)with results of a commonly used procedure (carbazole-borate reactivity after precipitation with cetylpyndinium chlo-ride). The present method was also used to assess metaboliccorrection in a patient with Hurler’s syndrome after treatmentby bone-marrow transplantation. This quantitative methodsurmounts the major technical problems of developing massscreening programs for infants, thus offering the potential forearlier diagnosis and treatment of mucopolysaccharidosisdiseases.

AddItIonal Keyphrases: neonate pediatric chemistryHurler’s syndrome . screening . bone-marrow transplanta-

tion

The mucopolysaccharidosis (MPS) storage diseases areunborn errors of glycosaminoglycan metabolism resultingfrom deficiencyof one of the at least 11 different enzymesinvolved in lysosomal catabolism of these sulfated hetero-polysaccharides.’ The consequent systemic accumulation ofheparan sulfate, dermatan sulfate, and (or) keratan sulfateis associated with specific clinical syndromes (Table 1),which recently have been reviewed (1-4). The mildest formsare characterized by debilitating skeletal abnormalities anddwarfism, progressive cardiopulmonary deterioration, anddeath during childhood or early adulthood. The more severeMPS diseases also result in progressive mental retardation.

MPS disease is first suspected between nine months andfour years of age when clinical abnormalities become mani-fest, and detection of increased glycosaminoglycans in urineconfirms the diagnosis. However, recent progress in thetreatment of these disorders (5-7) has demonstrated thatmuch earlier therapeutic intervention is crucial, thus pro-viding a strong motivation for presymptomatic diagnosis bymeans of routine mass screening of all newborn infants.

Current test methods used for diagnosis of these disorders

Institute of Human Genetics and Department of Pediatrics,University of Minnesota Medical School, 420 Delaware St., S.E.,Minneapolis, MN 55455; and Variety Club Children’s Hospital,Harvard St. and East River Road, Minneapolis, MN 55455.

1 Nonstandard abbreviations: DMB, 1,9-dimethylmethylene blue;GAG, glycosaminoglycan; MPS, mucopolysaccharidosis.

Received September 7, 1988; accepted December 8, 1988.

are technically cumbersome, labor-intensive, and requirelarge urine specimens (e.g., 2-10 mL). Furthermore, exist-ing methods depend on isolation of glycosaminoglycans byprecipitation with quaternary ammonium salts such ascetylpyridiniuni chloride (8, 9) or on separation techniquessuch as electrophoresis (10) or HPLC (11).

Some abbreviated “screening tests” have also been de-scribed, but these have been limited to tests that areapplicable to the relatively small number of children pre-senting with clinical features of MPS disease, and eventhese abbreviated tests are not practicable for mass screen-ing. In studies evaluating such methods only a few thou-sands of tests were done in each case (12, 13), typicallyduring severalyears. The tests are associated with relative-ly high false-positive and false-negative rates (14). For thesereasons, large-scale presymptomatic diagnosis of MPS din-eases has not been attempted.

To surmount these problems, we examined the potentialfor developing a direct dye-binding assay for quantifyingurinary glycosaminoglycan that involves no preparatoryisolation procedures. By exploiting the metachromatic prop-erties of the histochemical dye 1,9-dimethylmethylene blue(DMB), sulfated glycosaminoglycans can be directly mea-sured in very small urine specimens without cumbersomeseparatory techniques.

MaterIals and Methods

DMB dye reagent. For analysis of fresh or frozen-storedurine specimens, a stock solution containing 0.35 mmol of1,9-dimethylmethylene blue chloride (no. 03610-1; Poly-sciences, Inc., Warrington, PA) per liter, the “lOx stocksolution,” was made by dissolving 122 mg of dye in 10 mL of95% ethanol, which was then diluted to 1 L with sodiumformate buffer (pH 3.5, 0.2 mmol/L). This lOx stock solu-tion, stored in an amber-colored bottle at room temperature,could be used for up to two months. For analysis of referencesolutions or urine specimens, we prepared a 35 Hmol/L “lx”dye solution on the day of testing by appropriately dilutingthe lOx stock solution with the sodium formate buffer.

Collection of urine specimens. Urine specimens, collectedfrom normal individuals or patients with MPS, were eitheranalyzed promptly or stored at -70 #{176}Cuntil assay.

Glycosaminoglycan standards. Reference solutions wereprepared from heparan sulfate (from bovine kidney, no.H9637), keratan sulfate (from bovine cornea, no. K3001),chondroitin sulfate type A (from whale cartilage, no.C4134), dermatan sulfate (i.e., chondroitin sulfate type B,from porcine skin; no. C4259), and chondroitin sulfate typeC (from shark cartilage, no. C4384), all obtained from SigmaChemical Co., St. Louis, MO.

DMB dye-binding assay for glycosaminoglycan. For thestandard assay, 40 iL of sample (urine specimen fromnormal individuals, diluted urine specimen from patientswith Hurler’s or Hunter’s syndrome, or glycosaxninoglycanstandard) was mixed with 1.0 mL of 35 HmoI/L “lx” DMBreagent in plastic semi-micro spectrophotometer cuvettes(no. 2410; Stockwell Scientific, Walnut, CA 91789). Absor-

Detective enzyme

a-L-IdurOnidase

Table 1. BIochemical Nosology of the Mucopoiysaccharldosls DIseases (1-4)

CLINICALCHEMISTRY, Vol. 35, No. 3, 1989 375

Name

Mucopolysaccharidosistype!Hurler’ssyndrome,Scheie’s

syndrome, a Hurler-Scheiesyndrome,and variants

Mucopolysaccharldosis type IIHunter’ssyndrome,mildand

severe formsMucopo!ysaccharidosis type I!!

Sanfilippo’ssyndrometype ASanfilippo’ssyndrometype BSanfilippo’s syndrome type C

Sanfilippo’ssyndrometype DMucopo!ysaccharidosis type IV

Morquio’ssyndrometype A

Morquio’ssyndrometype BMorquio’ssyndrometype C

Mucopolysaceharidosis type VIMaroteaux-Lamysyndrome

Mucopolysacchandosis type VI!Sly’ssyndrome

Iduronatesulfatesulfatase

HeparansulfatesulfamidaseN-Acetyl-a-glucosaminidaseCoA:a-glucosaminide-N-acetyl-

transf eraseN-Acetyl-glucosamine-6-sulfate sulfatase

Galactosamine-N-acetyl-6-sulfatesulfatase

fl-Galactosidase(Unknown)

N-Acetyl-galactosamine-4-sulfatesulfatase

-Glucuronidase

Glycosaminoglycanspeciesin urine

Heparan sulfate, dermatansulfate

Heparan sulfate, dermatansulfate

Heparan sulfateHeparan sulfateHeparan sulfate

Heparan sulfate

Keratansulfate

Keratansulfate

Keratan sulfate

Dermatan sulfate

Heparan sulfate, dermatansulfate

Previously mucopolysacchandosistype V.

bance at 535 nm was measured within 30 mm in a BeckmanDU-8 or DU-5O spectrophotometer and compared with thatof appropriate standard solutions of chondroitin-6-sulfate.Color was assessed visually under standard indoor fluores-cent ceiling lighting.

Carbazole-borate assay for glycosaminoglycan. For com-parison, urinary glycosaminoglycan was quantified by themethod of Bitter and Muir (8) as modified by Di Ferrante(9). A lO-mL aliquot of urine was neutralized with concen-trated HC1 or 5 moIJL NaOH, precipitated overnight at 4#{176}Cwith cetylpyridinium chloride (final concentration 2.5 g/L),and then assayed for uronic acid with the carbazole-boratereagent. The absorbance at 522 nm was measured andcompared with that of reference solutions of (3-glucuronolac-tone (no. G8875; Sigma). Glycosaminoglycan content wasexpressed as grams of uronic acid per liter of urine.

Creatinine assay. Urinary creatinine was measured by amodification of the method of Folin and Wu (15) by mixing50 j.iL of urine and 200 pL of distilled water with 1.0 mL of afivefold dilution of saturated picric acid (no. 925-4(); Sigma)and 1.0 mL of a 7.5 g/L solution of sodium hydroxide. Afterreaction for 20 miii, the absorbance at 535 nm was mea-sured and compared with that for creatinine referencesolutions of 10, 30, 100, and 150 mg/L concentrations (no.925-11 and 925-15; Sigma).

Data management and statistical methods. To computeresults, we programmed Lotus 1-2-3 version 2A (LotusDevelopment Corp., Cambridge, MA) to calculate the slopeand intercept of the linear portion of standard curves forglycosaminoglycan and creatinine by regression analysis, tocalculate glycosaminoglycan and creatinune concentrationsof test samples, and to calculate urinary glycosaminoglycanas the ratio glycosaminoglycan/creatinine (milligrams ofglycosaminoglycan per gram of creatinune). Lotus 1-2-3 andCricket Graph version 1.2 (Cricket Software, Malvern, PA)were used for graphic illustrations and calculation of corre-lation coefficients.

Pharmaceutical formulations. To study potential interfer-

ence of pharmaceutical coloring agents in the test system,we obtained commonly prescribed pediatric preparations forstudy, specifically Augmentin (Beecham Laboratories, Bris-tol, TN), Cechlor (Eli Lilly, Indianapolis, IN), erythromycin(Abbott Laboratories, North Chicago, IL), furosemide (Ly-phomed Inc., Melrose Park, IL), morphine sulfate (Win-throp-Breon, Des Plaines, IL), Pediazole (Ross Laboratories,Columbus, OH), penicillin (Pflzerpen; Pfizer Inc., New York,NY), Rondec syrup (Ross Laboratories), Septra (BurroughsWellcome, Research Thangle Park, NC), Tavist (Sandoz,Parsippany, NJ), Trianunic Cold Syrup (Sandoz), Triaminic-DM (Sandoz), and Tylenol drops (McNeilab, Fort Washing-ton, PA). Also evaluated were heparin from porcine bowel(1000 USP units/mL; Elkuns-Sinn, Cherry Hill, NJ) andbovine serum albumin (Sigma).

Results

Standard Assay Conditions

Optimal assay conditions were determined in a series ofexperiments to study the absorption spectrum of DMB andglycosaminoglycan-DMB (GAG-DMB) complexes undervarious conditions of glycosaminoglycan concentration, pH,and species of glycosaminoglycan.

Absorption spectnim of DMB and GAG-DMB complexes.To determine the absorption spectrum of DMB and GAG-DMB complexes, various concentrations of heparan sulfatewere added to the standard reaction mixture at pH 3.5 andscanned over the range of 400-700 nm after blanking thespectrophotometer against a cuvette containing 0.2 mol/Lformate buffer without dye. Identical spectra were obtainedin solutions having a pH of 3.0, 4.0, 4.5, or 5.0 (data notshown).

Saturation of GAG-DMB complex formation. As an initialmeans of evaluating the potential useful range of glycos-aininoglycan concentration, the color and absorbance ofvarious concentrations of heparan sulfate were determined.The visible color changes and increasing absorbance were

040

0.35

0.30a,

C)

0.25a0

0.20

0.15

0.10

0.05

.S. S #{149}

:1o #{149}Heparan Sulfate

S Keratan Sulfate

#{163}Chondroitin-4-Sultate

0 Dermatan Sulfate0 Chondroitin-6-Sulfate

00 j 0 0

0.4

0.3

0.2

0.1

C)C‘a.00a.0

0

0 0

y = 1.0020e-3 + 2.2507e-3x R2 0.9862 3 4

0 200 400 600 800- 1000 1200

376 CLINICALCHEMISTRY,Vol.35, No.3, 1989

observed within the first few seconds after heparan sulfatewas mixed with DMB reagent. While reference solutionswith concentrations of 60 tg/mL or less retained the bluecolor of unreacted DMB, solutions with 70 to 90 pg/mLappeared lavender, and samples with 100 to 1000 ug/mLwere brilliant pink and were prone to precipitate over time.Increasing heparan sulfate concentrations above approxi-mately 200 jzg/mL resulted in no additional increase inabsorbance, the maximum being about 0.35 A.

Specificity of GAG-DMB complex formation. Other gly-cosaminoglycans were then similarly studied. The diagnos-tically important sulfated glycosaminoglycans (heparan sul-fate, keratan sulfate, chondroitin-4-sulfate, dermatan sul-fate, and chondroitun-6-sulfate) exhibited the same reactionwith the dye. When presented as a semilogarithmic plot,absorbance was seen as a sigmoidal function of glycosamuno-glycan concentration (Figure 1), with the same maximalabsorbance being reached by glycosaminoglycan concentra-tions of 200 mg/L or more. Much higher glycosaminoglycanconcentrations often resulted in a visible GAG-DMB precipi-tate and a corresponding decrease in absorbance (data notshown).

Linear range for routine &tenninations. The relationshipbetween glycosaminoglycan concentration and absorbanceof GAG-DMB complexes was examined in the range ofconcentrations between 1 and 1000 ig/mL. For each of thesulfated glycosaminoglycans, the absorbance of the GAG-DMB complex was, up to a point, linearly proportional toglycosamunoglycan concentration (e.g., heparan sulfate, Fig-ure 2). However, absorbances of <0.025 A were consideredunreliable, sample-to-sample variation being considerableowing to the limitations of instrumentation. Therefore,glycosaminoglycan concentration can be determined by di-rect linear extrapolation from absorbance over the approxi-mate range 10 to 150 zg/mL. By this method, DMB exhibit-ed the same reactivity with each of the different sulfatedglycosaminoglycans. This observation contrasts with theprevious observation (16) that DMB is less reactive withkeratan sulfate.

045

0.00

Glycosaminoglycan(Log mg/L)

Fig. 1. Congruentreactivity of glycosaminoglycanspecies with 1,9-dimethylmethylene blue dyeAs measuredby change in absorbance at535 nm,theDMBreagentreactedwitheach of the pathologicallysignificant glycosaminoglycans (heparan sulfate,keratan sulfate, chondroitin-4-sulfate, dermatan sulfate, chrondroltln-6-sulfate),GAG-OMBcomplex formation becoming saturated at approdmately200 tg ofglyoosaminoglycan per milliliter

For routine analysis of urine specimens, a series ofreference solutions of chondroitin-6-sulfate (25, 50, 75,350 g/mL) was treated in parallel with test samples, andthe upper limit of linearity was re-assessed in each seriesbased on this standard curve. Concentrations exceeding theupper limit, typically 0.30 A, could be exactly quantified bytesting an appropriate dilution of the original specimen.

Theoretical Considerations Regarding Application

Comparison with carbazole-borate/cetylpyridinium chlo-ride methodology. The direct DMB method was comparedwith the conventional procedure of Bitter and Muir (8) asmodified by Di Ferrante (9). For this comparison, urinesamples extending over the range of normal and pathologi-cal glycosaminoglycan concentrations were analyzed byboth methods. Six urine samples were collected from each offour normal individuals (n = 24; “low glycosaminoglycan”)and from each of four MPS patients (three with Hurler’ssyndrome, and one with Hunter’s syndrome, n = 24; “highglycosarninoglycan”). We could assay a batch of 10 to 30samples and calculate the glycosaniinoglycanlcreatimne ra-tio by the present method in less than 2 h. Less than 0.1 mLof urine is required for determination of both glycosamino-glycan and creatinine. In contrast, the conventional two-daycetylpyridinium chloride-carbazole--borate method requiresovernight precipitation of duplicate urine specimens (10 mLeach), followed by sequential washing of precipitated glycos-aminoglycan in cold ethanol, drying, and rehydration of thespecimen, and finally assay of uronic acid content. Resultsfor 48 urine specimens are represented graphically (Figure3). The present method correctly identified all 24 MPSspecimens on the basis of high glycosaminoglycan (range:117 to 813 mg of glycosaminoglycan per gram of creatinine)and discriminated the 24 specimens from normal adults(range: 6.3-19.0 mg/g). Linear regression analysis indicateda correlation coefficient of 0.764.

Potential interfering substances. Potential interferencefrom some pharmaceutical formulations and protein inurine was assessed by determining the effect of each in thestandard assay reaction mixture (Table 2). For each sub-stance, 40 pL of the specified material was added at theindicated concentration in place of the usual urine test

Heparan Sulfate (mg/L)

Fig.2. Linear rangeof glycosaminoglycan-DMB complexformationAs shown for heparan sulfate, glycosaminoglycan concentration can be estimat-ed by linear extrapolation from absorbance up to approxImately150 gImL

a,C.2 :

013

0 200 400 600 800 1000 1200 1400

Substanc, or pharmaceutIcalfermulatlon

Albumin, bovine, 1 mg!mL

Amoxicillin, 25 mg!mL, &potassiumclavulanate,6.25mg!mL (Augmentin 125)

Cefaclor 25, mg/mL (Ceclor)

Erythromycin, 50 mg/mL

Furosemide,10 mg!mL

Hepann, porcine, 1000 USPunits/mL

Morphinesulfate,2 mg!mL

Observedcobra

PinkPinkLavender

LavenderLavender

Brown

Lavender

Brown

1:101:100

1:101:100

1:101:100

1:101:100

1:101:100

1:101:100

1:101:100

1:101:100

1:101:100

0.010.000.00

1.250.830.03

-0.02-0.01

0.01

0.000.000.00

-0.08-0.09

0.01

0.000.000.00

0.090.000.00

0.140.020.01

0.07-0.01

0.00

Table 2. Evaluation of Potential Interfering Substances

CLINICAL CHEMISTRY, Vol. 35, No. 3, 1989 377

CPC/Carbazole Method(mg Uronic Acid/g Crealinine)

Fig. 3. Conipaflson of methodologies (direct 0MB vs cetylpyndiniumctiloride/carbazole-borate) for quantification of urinary glycosamino-glycan excretionThe directDMBtest correctlydifferentiated24 MPS patients’specimenson thebasis of high glycosaminoglycan(range: 117-813 mg glycosaminoglycanpergramcreatinine) from 24 specImens fromnormaladults (lowglycosaminoglycan,range:6.3-19.0 mg/g).Quantitativeresultswere moderately correlated (r = 0.76)with results of a standard method (cetlpyrldinium chloride precipitableuronicacid as measured withthe carbazole-borate reagent)

sample. Albumin was nonreactive with dye at a concentra-tion of 1 mg/mL. In contrast, the anticoagulant heparin, asulfated glycosaminoglycan, was found to be highly reactivein the assay. Other drugs likely to be administered toinfants produced no interference except when formulationscontaining artificial coloring agents were added to thereaction at full strength.

Clinical application. Five 1-mL urine specimens wereobtained from a one-year-old girl with Hurler’s syndromeand, when tested by the direct DMB method, were found tohave pathognomonic concentrations of glycosaminoglycan(Figure 4). After bone-marrow transplantation from a nor-mal histocompatible donor, urinary glycosaminoglycan do-dined, reaching the lowest values 40-60 days after trans-plantation. Drugs administered to patients during bone-marrow transplantation and excreted in urine did notappear to interfere with quantification of urinary glycos-aminoglycan.

DIscussion

As originally recommended in the Scriver guidelines (17),institution of mass screening of newborns must be justifiedon the basis of several pragmatic criteria. Some degree ofsuccessful treatment of the MPS diseases has recently beenachieved (5-7). Practical, low-cost testing methodology hasbecome a paramount consideration. Despite numerous stud-ies describing putative “screening” tests for MPS diseases,no such test is routinely applied for mass screening of thenewborn population. Part of the failure to implement new-born screening can be ascribed to problems inherent inavailable testing methodologies. Existing tests are labor-intensive and thus prohibitively expensive, and many re-quire large sample volumes (2-10 mL), which cannot beroutinely collected from infants.

As one of the most broadly applied metabolic screeningprocedures, the “Berry spot test” (12, 18) has been widelyused for preliminary testing of children in whom the diagno-

DIlution Absorbancefactor’ (535 nm)b

1 0.02

1 0.271:10 0.091:100 0.02

1 0.291:10 0.071:100 0.02

1 0.001:10 0.011:100 0.01

1 0.001:10 0.001:100 0.00

1 0.451:10 0.301:100 0.17

Erythromycin ethylsuccinate,20 mg/mL, & sulfisoxazoleacetyl, 60 mg/mL (Pediazolesuspension)d

PenicillinG, 50 000 LiSPunits!mL (Pfizerpen)

Carbinoxaminemaleate,0.4mg/mL,& pseudoephedrineHCI, 6 mg/mL(Rondecsyrup)d

Trimethoprim, 16 mg/mL,&sulfamethoxazole,80 mg!mL(Septra)

Clemastine,25 ag!mL (Tavist)

PhenyipropanolamineHCI,625 ug!mL, & chlorphenira-mine maleate, 0.1 mg/mL(Triaminic)d

Phenylpropanolamine HCI,625 pg!mL, & dextromethor-phanHCI, 0.5 mg/mL(Trianlinic-DM)’

Acetaminophen, 100 mg/mL(Tylenol drops) (I

‘Pharmaceuticalformulationwas added full strength (1) or diluted(1:10 or1:100)indistilledwater.

#{176}Absorbanceat 535 nm was measuredafteradditionof 40 L of potentialinterfenngsubstance to 1.0 mL of 35 mol/L DMB reagentunder standardreaction conditions.

CBlueunlessotherwisenoted.a Pharmaceutical formulationscontainingan artificialcoloringagent:Aug-

mentin (Yellow no. 10), Ceclor (Blue no. 1 and Red no. 3), Pediazolesuspension(unspecified),Rondecsyrup (Red no. 33 and Yellow no. 6),Tnamlnic-DM(Blueno. 1 and Red no. 40), Triaminic(Yellowno. 6). Tylenoldrops(Yellowno 6).

a,Qa,.2’ C

o oC.,-.->.

.0C

25.

800

600

400

200

0-20 0 20 40 60

1000

378 CLINICALCHEMISTRY, Vol. 35, No. 3, 1989

Post.Transplantation Period (Days)

Fig. 4. Unnatyglycosaminoglycan after bone-marrow transplantationfor Hurler’s syndromeIn five urine specimens obtainedbefore transplantation, tie direct 0MB testconfirmedhigh glycosaminoglycan excretionby a patient with mucopolysacchari-dosis type I (Hurler’s syndrome). After bone-marrow transplantation from anormalhistocompatibledonor,unnaly glycosaminoglycandeclined,and was low40-60 days after transplantation

sis of MPS disease is suspected. The Ames Company’s MPSpaper spot test, technically similar to the Berry spot test(19), has been applied in a semiquantitative manner (10).However, both of these methods are only semiquantitativeat best, and neither is readily automatable.

Dye-binding techniques have been attractive, particularlybecause of the sensitivity and potential for quantitativeresults. Among the available dye reagents, Alcian blue hasbeen used for direct quantification of glycosaminoglycan inurine (20), although this method has been criticized due todifficulty in harvesting finely dispersed precipitate andinterference from negatively charged molecules (14, 21).Alcian blue has also been used to quantify glycosaminogly-cans after separation by electrophoresis (22), although thistechnique is a 7-h procedure that requires boiling of precipi-tates formed with cetylpyridinium chloride.

The DMB dye reagent used in the present method wasfirst described as a histochemical stain by Taylor and Jeifree(23). It was subsequently applied to a colorimetric assay forsulfated glycosaininoglycans by Humbel and Etringer (24),who also suggested that DMB could be used in a simpleurine screening test for MPS diseases by exploiting thevisual color change. By their method, the color of DMBreagent added to urine specimens changed from blue to red-violet in the case of affected children. The test was reportedto have a very low false-positive rate, <0.5%, and the 16%false-negative rate was attributed to failure to detect onlycases of Morquio syndrome. In contrast to the originalreport, when this visual screening test was evaluated in ourlaboratory, there were many false-positiveand false-nega-tive results, making it unacceptable.

Farndale et al. (25) described a DMB method for quantify-ing glycosaminoglycan in cartilage tissue culture, findingthat GAG-DMB complexes could be stabilized against pre-cipitation by substituting formate buffer for dibasic citrate-phosphate. They noted that the DMB reagent was moresensitive than Alcian blue, and they also proposed that thespectrophotometric measurements could be automated, afeat subsequently accomplished by Sabiston et al. (26).

During the course of our work, Panin et al. (21) demon-

strated that DMB could be used to quantify glycosamino-glycan isolated from urine, but their method required largealiquots from 24-h urine specimens and the cumbersomeisolation procedure of overnight precipitation with cetylpy-ridinium chloride and washing with alcohol.

To exploit the sensitivity of a dye-based assay, we ex-plored the specificity of DMB for sulfated glycosaminogly-cans in urine. As developed here, with results expressed interms of urinary creatiine concentration, the direct DMBmethod is technically simple and therefore both rapid andinexpensive, with precisely quantitative results. Further-more, the very small sample requirement (<0.1 mL of urine)is appropriate to mass screening of neonates.

In looking for potential interferences, we found thatalbumin, at concentrations found in urine, is nonreactive

80 with the dye. Heparin, a sulfated glycosaminoglycan, ishighly reactive in the assay. However, newborn infants donot receive heparmn except in the setting of the newbornintensive-care unit. Other drugs likely to be administered toinfants did not interfere except for those containing artifi-cial coloring agents, and then only when added at fullstrength and not at concentrations expected in urine.

The direct DMB method provides a quantitative test foridentifying excessive excretion of the abnormal metabolitespathognomomc of MPS disease. The precisely quantitativenature of the test gives the method additional application inmonitoring the response to treatment such as bone-marrowtransplantation. To merit widespread implementation formass screening, the error rates (false positive, false nega-tive) must be relatively small (17), a major problem ofexisting MPS tests (14). From preliminary results (27), wepredict that studies of larger populations of normal infantsand MPS patients will permit assignment of limits associat-ed with acceptable error rates.

In summary, this new direct method of quantifying exces-sive excretion of sulfated glycosaminoglycans is ideallysuited to mass screening programs for MPS diseases, and itcircumvents the problems of existing methodologies. Itrequires only a few drops of urine, and many specimens canbe assayed in less than 2 h with standard laboratoryinstrumentation. As a basis for discriminating normal andpathological values, urinary glycosaminoglycan excretioncan be expressed in terms of urinary creatinine. Test resultsof the direct DMB method correlate with those of thestandard cetylpyridinium chloride/carbazole-borate meth-od. Based on these observations, ongoing studies are direct-ed toward determining normal and pathological values inrepresentative populations of age-matched normal and af-fected children (27).

This work was supported by grants from the Preston Arnold MPSResearch Fund, from the National MPS Society through a contribu-tion by the family of Kirby Lastinger, and by grants from the BoneMarrow Transplantation Research Fund, the Minnesota MedicalFoundation, and the Human Growth Foundation. We thank Dr. P.Jean Henslee and Dr. Ronald Whitley, University of Kentucky,Lexington, for providing urine specimens from the patient withHurler’s syndrome who was undergoing bone-marrow transplanta-tion.

References1. Spranger J. The mucopolysaccharidoses. In: Emery AEH, Ri-mom DL, eds. Principles and practice of medical genetics. NewYork: Churchill Livingstone, 1983:1339-47.2. McKusick VA, Neufeld EF. The mucopolysaccharidosisstoragediseases.In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Gold-

CLINICALCHEMISTRY, Vol. 35, No. 3, 1989 379

stein JL, Brown MS, eds. The metabolic basis of inherited disease,5th ed. New York: McGraw-Hill, 1983:751-77.3. Muenzer J. Mucopolysacchandoses [Review]. Adv Pediatr1986;33:269-302.4. SprangerJ. Mini review: inborn errors of complex carbohydratemetabolism. Am J Med Genet 1987;28:489-99.5. HobbsJR, Hugh-JonesK, Barrett AJ, et al. Reversal of clinicalfeatures of Hurler’s diseaseand biochemical improvement aftertreatment by bone marrow transplantation. Lancet 1981;ii:709-12.6. Krivit W, Pierpont ME, Ayaz K, et al. Bone marrow transplanta-tion in the Maroteaux-Lamy syndrome (mucopolysaceharidosistype VI): biochemical and clinical status 24 months after transplan-tation. N Engl J Med 1984;311:1606-11.

7. Whitley CB, Ramsay NKC, Kersey JH, Krivit W. Bone marrowtransplantation for Hurler syndrome: assessment of metaboliccorrection. Birth Defects 1986;22:7-24.8. Bitter T, Muir HM. A modified urcmc acid carbazole reaction.Anal Biochem 1962;4:330-4.9. Di Ferrante NM. The measurement of urinary mucopolysaccha-rides. Anal Biochem 1967;21:98-106.

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