Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease.

Marked elevation of plasma chitotriosidase

activity. A novel hallmark of Gaucher disease.

C E Hollak, … , M H van Oers, J M Aerts

J Clin Invest. 1994;

93(3)

:1288-1292.

https://doi.org/10.1172/JCI117084

.

Gaucher disease (GD; glucosylceramidosis) is caused by a deficient activity of the enzyme

glucocerebrosidase (GC). Clinical manifestations are highly variable and cannot be

predicted accurately on the basis of the properties of mutant GC. Analysis of secondary

abnormalities, such as elevated plasma levels of some hydrolases, may help to increase

insight into the complicated pathophysiology of the disease and could also provide useful

disease markers. The recent availability of enzyme supplementation therapy for GD

increases the need for markers as early predictors of the efficacy of treatment. We report the

finding of a very marked increase in chitotrisidase activity in plasma of 30 of 32 symptomatic

type 1 GD patients studied: the median activity being > 600 times the median value in

plasma of healthy volunteers. In three GC-deficient individuals without clinical symptoms,

only slight increases were noted. Chitotriosidase activity was absent in plasma of three

control subjects and two patients. During enzyme supplementation therapy, chitotriosidase

activity declined dramatically. We conclude that plasma chitotriosidase levels can serve as

a new diagnostic hallmark of GD and should prove to be useful in assessing whether

clinical manifestations of GD are present and for monitoring the efficacy of therapeutic

intervention.

Research Article

Rapid Publication

Marked Elevation of Plasma Chitotriosidase Activity

A Novel Hallmark of Gaucher Disease

Carla E. M.Hollak,**Sonja_vanWeely,*Marinus H.J._vanOers,*andJohannesM. F. G. Aerts*

Departments of *Biochemistry and $Hematology, Academic Medical Centre, 1105 AZ Amsterdam, TheNetherlands

Abstract

Gaucher disease (GD;glucosylceramidosis)iscaused by a defi-cientactivity of the enzymeglucocerebrosidase (GC).Clinical manifestations are highly variable and cannot be predicted accu-rately on the basis of the properties of mutant GC. Analysis of secondary abnormalities, such as elevated plasma levels of somehydrolases, may help to increase insight into the compli-cated pathophysiology of the disease and could also provide

useful diseasemarkers. The recent availability of enzyme sup-plementation therapy for GD increases the need for markers as earlypredictors of the efficacy of treatment. We report the

findingof a very marked increase inchitotriosidaseactivity in plasma of 30 of 32 symptomatic type 1 GD patients studied: the medianactivity being > 600 times the median value in plasma

ofhealthy volunteers. In three GC-deficient individuals without

clinicalsymptoms, only slight increases were noted.

Chitotrio-sidase activitywas absent in plasma of three control subjects andtwopatients. During enzyme supplementation therapy,

chi-totriosidase activity declined dramatically. We conclude that plasma chitotriosidase levels can serve as a new diagnostic hall-mark of GD and should prove to be useful in assessing whether clinicalmanifestations of GD are present and for monitoring theefficacyoftherapeutic intervention. (J.Clin.Invest. 1994. 93:1288-1292.) Key words: Gaucher disease * glucosylcerami-dosis* chitotriosidase * enzyme therapy * acidphosphatase

Introduction

Gaucher disease

(GD)'

is characterizedby accumulation of

glucosylceramide (glucocerebroside)in the lysosomes of mac-rophages. Thisisdue to aninherited deficiencyin theactivity

ofglucocerebrosidase (GC),alysosomalhydrolase ( 1 ).

Accu-mulationof lipid-ladenmacrophages results in hepatospleno-megaly, bone lesions, and, less commonly, inneurological

ab-Addresscorrespondence to Dr. JohannesM.F. G.Aerts, Department ofBiochemistry, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Receivedfor publication 4 October 1993 and in revisedform 3 De-cember1993.

1.Abbreviations used in this paper: AP, acid phosphatase 5b; GC, glu-cocerebrosidase; GD, Gaucher disease; MU, methylumbelliferyl; SSI, severity scoring index.

normalities. For the mostfrequent nonneuronopathic

pheno-type(type1)nostrict correlationhas been established between

mutant genotypes, residual GCactivity,and clinicalexpression ofthedisease ( 1, 2).Even amongsiblings sharingthe same GC

genotype astrikingdifferencein clinicalsymptoms mayoccur.

It has beenclaimedthatoneof themostfrequentmutant GC

genotypes,homozygosityfor theN370Smutation,is associated

with mild disease (3, 4). However, with this genotype,

com-pletelyasymptomatic elderlyindividualsas well asseverely

af-fectedyoungpatientshave beendescribed. The limited

correla-tionbetweengenotypeandphenotypesuggestsamore

compli-cated pathophysiology of the disease and hampers genetic counselling.This has stimulated interestinsecondary biochem-ical abnormalities associated with clinical manifestations of GD. Elevatedplasma levels ofacid

phosphatase

Sb (AP)are

traditionally consideredtobe themost prominent secondary biochemical abnormality ( 1). Moderate increases inplasma

levels of

f3-hexosaminidase, angiotensin

converting enzyme, andlysozymehavealso beendescribed

(1). Recently

enzyme

supplementation

therapy with alglucerase has become avail-able.Since algluceraseisextremely costlyand clinical

improve-mentduringenzyme

supplementation

therapy isslow

(usually

requiringatleast 3-6 moof treatment) (5, 6),the searchfor secondary biochemicalabnormalitiesas

possible

early indica-torsofresponse to treatment has becomeanissue ofincreasing importance.

Here wereporton an extremeelevationinchitotriosidase

activity

in the plasma oftype 1 GD

patients.

The increased plasma levelsof this novelenzymewerestudiedinrelationto

those ofAPand

lysozyme.

Its

possible

sourcewas

investigated.

Theclinicalsignificance ofthese

findings

withrespectto

diag-nosis, disease severity, and the

monitoring

ofthe

efficacy

of

enzyme

supplementation

therapy is discussed.

Methods

AllGDpatientsstudied ( 16 males and 16females;3-72 yrold)were

known to useither bycontactwith the Netherlands GaucherSocietyor

by referraltothe Academic Medical Center.Thediagnosisof GDwas

basedondeficientglucocerebrosidase activityinleucocytes and/or

ur-inesamples (7, 8). The clinical manifestations of 25patients (11males and14females; 15-72 yrold)wereclassifiedasbeing mild, moderate,

or severebyusingthe modifiedseverity scoringindex(SSI) (9),which is basedon an assessmentof theextentofliver,spleen, and bone in-volvementandtheseverityofpancytopenia.Using theSSI,mild dis-easewaspresent in7 patients, moderatedisease in 12patients, and severediseasein6patients.Nopatienthadacutemanifestations ofthe disease.Duringfamily screening,three other individuals(onemaleand twofemales;71, 78, and96 yrold)werefoundtohavedeficient GC

activity,butnoclinicalexpressionof GD.Thecontrolpopulation

con-1288 Hollaketal. J.Clin. Invest.

©D

TheAmerican Society for Clinical Investigation, Inc. 0021-9738/94/03/1288/05 $2.00

sisted of 50 healthy volunteers (30 males and 20 females; 23-56 yr

old), allof whom were found to have normal GC activity.

EDTA plasma samples were obtained from freshly drawn blood andimmediately stored at -20'C.

Four patients were studied during therapy with intravenous alglu-cerase(Ceredase", Genzyme Corp., Cambridge, MA) at a dose of 4 U/kg, threetimesaweek.

Chitotriosidase activity was measured by incubating 5

_,d

of EDTA plasma with 100ulof 0.022 mM

4-methylumbelliferyl-fl-D-NN,N'-triacetylchitotriose (4 MU-chitotrioside; Sigma Chemical Co., St. Louis, MO) as substrate in citrate/phosphate buffer(0.1/0.2 M), pH 5.2, at370C.In GDpatients, samples were diluted50Xin demineral-ized water before incubation. After 15 min the reaction was stopped with 2 ml of 0.3 M glycine/NaOH buffer, pH 10.6. Fluorescent 4-methylumbelliferone was measured with a fluorimeter (Perkin-Elmer Corp., Norwalk, CT)at445nm.Chitotriosidase activity in the super-natantof cultured cells was measured by incubating 10Mlof the super-natantwith 100 Ml of substrate mixture for 30 min. The enzyme activi-tieswerelinear with timeof incubation and amount of enzyme (data not shown).

Lysozymeactivity towards cell wall suspension of Micrococcus

ly-sodeikticus (Sigma Chemical Co.) was determined as described by

Morsky (10).

Acid phosphatase activity was measured using 4-methylumbelli-feryl phosphateassubstrate in the presenceof 3Mmercaptoethanol as described by Chambersetal.(11).

Immunoprecipitation studies were performed with a rabbit anti-serum tochitotriosidase (obtained after immunization ofarabbit with thepurified enzyme) or withanti-(lysozyme)antibodies (Zymed Labs., Inc.,San Francisco, CA). Chitotriosidasewaspurified from the soluble fractionof adetergent-freehomogenate of type 1 Gaucher spleen bya sequence ofchromatographic steps, includingchromatofocussing,gel

filtration,andisoelectricfocussing.Antibodieswerecoupledtoprotein

A-Sepharose4Bbeads, andan excessof the immobilized antibodies was incubated with plasmafor 1hat room temperature. After centrifu-gation, enzyme activities in the supernatants were measuredas de-scribed above.

Monocyteswerepreparedasfollows: mononuclear cellswere iso-lated from citrated blood, diluted 1:1 with PBS, by Percolldensity

gradientcentrifugation( 1.077g/cm3, 18 min, 2,000 rpm). Aftertwo washing steps, one in PBS supplemented with 0.38% sodium citrate andonein autologous plasma, cellswereresuspended in Percoll ( 1.063

g/cm3)andcarefullylayeredonPercoll( 1.074g/cm3).After centrifu-gation ( 18 minat2,000 rpm), monocyteswerecollectedfromaband ontop of thegradient.The cellswerewashed andresuspendedin cul-turemedium (RPMI 1640supplemented withL-glutamineand 10% humanABserum). The monocytepreparationshadapurity exceeding

85%asjudgedby Giemsastaining. Monocyteswerecultured inplastic

petridishesat aconcentration of 4X105 cells/mlin 10 mlof culture medium.During20d of culture, 1.8mlof the culture supernatantwas collecteddailyandreplaced by fresh medium. Aftercentrifugation,the supernatantwasimmediatelyfrozenat-20°C.

Results

Fig. 1shows theplasmalevelsof

chitotriosidase, lysozyme,

and

APin 32 untreated

symptomatic

GD

patients

(III), 3

asymp-tomatic GC-deficient individuals

(II),

and50

healthy

controls

(I).Chitotriosidaseactivitywas

strikingly

increasedin30GD

patients (median, 12,824; range,

3,122-65,349 nmol/ml

per h)ascompared withasymptomatic GC-deficient individuals (90, 166,and 195 nmol/mlperh)andhealthycontrols (me-dian, 20;range,4-76

nmol/ml

per

h)

(Fig.

1A).Inthe30 GD

patients chitotriosidasewasincreased2

100-fold,

themedian

valuebeing>600timesthemedian valueofthe control sub-jects. However,intwoGD

patients,

chitotriosidaseactivitywas

almostabsent

(2

and3

_nmol/ml

_per

_h).

_Mixing

_experiments

indicated that the

plasma samples

ofthese

patients

contained

noinhibitors ofthe enzyme. In thethree

asymptomatic

GC-de-ficient individuals the elevation with

_respect

to the median controlvaluewas

only 9.7, 4.5,

and 8.3 times.

Since it isknownthat the 4MU-chitotriosidesubstratecan

alsobe

_{hydrolyzed by lysozyme}

_{( 12),}

thecontribution ofthis

enzymetothe observedchitotriosidase

_activity

wasassessed

by

the measurementofthe

activity

of

lysozyme,

using

a

suspen-sion ofcell walls ofM.

_{lysodeikticus}

as substrate.

_Lysozyme

activity,

measured in 27 ofthe 32 GD

_patients,

was

only

slightly

increased

(median,

4.1;

range, 1.3-9.5

U/,ul)

as

com-pared

with thecontrols

_(median,

1.7;

range,0.7-2.9

U/,ul)

and the

asymptomatic

individuals

₍

_{1.2, 1.7,}

and3.5

_U/,ql).

Interest-ingly,

in both GD

_patients

with low chitotriosidase

activity,

elevations in

lysozyme

activity

(4.1

and5.0

_U/Mgl)

weresimilar

tothose in the otherGD

_{patients. Comparison}

of

chitotriosi-dase and

lysozyme

activitiesin the30GD

patients

revealedno

correlation

_(r

=_0.

15),

indicating

that theobserved

chitotriosi-dase

_activity

can notbe

explained by lysozyme activity.

More

importantly,

chitotriosidase

activity

in GD

_{plasma samples}

proved

tobe almost

completely

precipitable

with

anti-(chito-triosidase)

antiserum,

butnot atallwith

_{anti-(lysozyme)}

anti-bodies. Onthe other

hand,

plasma

lysozyme

couldnotbe

im-munoprecipitated

with

_{anti-(chitotriosidase)}

antiserum.Inthe

twoGD

_patients

with verylow chitotriosidase

activities,

im-munoprecipitation

with

anti-(lysozyme)

antibodies showed

that the lowresidual

_activity

present

was

entirely

dueto

lyso-zyme. Thesame

phenomenon

wasfoundin the threecontrol

subjects

with

_relatively

lowchitotriosidase activities

(4,

4,

and 6

_nmol/ml

per

h) (data

not

_shown).

AP

_activity

waselevatedtoavariableextentin

_plasma

ofall

GD

patients (median, 2,590;

range,

452-9,785

nmol/ml

perh

in the30

patients

with

high

chitotriosidase

activity;

1,159

and

4,764

nmol/ml

perhin the 2

patients

withlowchitotriosidase

activity),

when

_compared

withcontrols

_(median,

202;

range,

94-342

_nmol/ml

per

h)

and

asymptomatic

individuals

(100,

130,

and177

_nmol/ml

_per

_h).

InGD

_patients,

theelevationsin APactivitieswere

clearly

farless

prominent

than those in

chi-totriosidase activities

_(Fig.

1,

A and

_B).

InGD

patients,

the

_macrophages

are a

likely

cellularsource

ofbiochemical abnormalities

(

1).

We therefore studied the

production

andsecretion of chitotriosidase

activity during

dif-ferentiation of cultured

monocytes

into

macrophages. During

the first 5 dofculture of

_purified

monocytes,

chitotriosidase

activity

wasfoundtobeabsent both in the cells and superna-tant. After

_{morphological}

differentiation of

monocytes

into

macrophages,

the cells

_began

to

_produce

andsecrete

increasing

amountsof chitotriosidase

(Fig.

2).

APwasalso

produced

and

secreted after5 dof culture

_(Fig.

2) (see

reference

_13).

Wedidnotfindacorrelationbetweenchitotriosidase levels

and the

severity

of clinical

_{manifestations,}

asassessed

by

the use of the SSI

(data

not

_shown).

However,

in

asymptomatic

GC-deficient individuals

only

mildelevationswerefound.

Like-wise,

chitotriosidase levels did not correlatewiththe mutant

GC

_genotype

in the GD

_patients

_(data

not

shown).

Four

patients

were treated

by

enzyme

supplementation

therapy

for _{2 1 yr.} After 3-6 mo, clinical

improvement

was

apparent

with_respectto

_{hematological}

markers andreduction

in

_organomegaly

_{( 14).}

_Fig.

3,

A-D,

showsthatchitotriosidase

levels

_rapidly

declined

during

enzyme

supplementation

100.000 10.000 *5- 1.000 o *

O0

U) I_ 100 o .! E_{= t}

A

I

0

0

0

2

II n=50 n=3 10 _I > 8 Eo o = _)-1.

B

0 0 III n=32 0

ii

a A A a A A A Ao A

I

A n=50 0 A0 0 A

II

11 n=3 III n=32

Discussion

Chitotriosidase activityin plasma samplesofuntreated type 1 GD patients was found to be strikingly elevated. Moreover,

chitotriosidaseactivity began to decline rapidly after the initia-tionofenzymesupplementation therapy. Previously, the occur-renceofachitotetraosidase activity in normal human plasma hasbeen reported ( 15 ). The chitotriosidase described here re-sembles the plasma chitotetraosidase with respect to the

appar-entbasic isoelectric pointandnonbinding to the lectin

conca-navalinA.Itistherefore possible that chitotriosidase and

chito-tetraosidase activitiesare due to asingleenzyme. An increase ineither chitotriosidaseorchitotetraosidase activities in

associ-ation with pathological conditionshas sofar not been reported.

.10.000 .5 -0) ._ 1.000 a 0 4. -In -oE a 2 E C - 100 °

Figure1.Levelsofplasma chitotrio-sidase (A), andlysozymeandAP

activity(B) in controls(I, n=50), asymptomatic GC-deficient individ-uals(II,n=3), andsymptomatic

GDpatients (III,n=32).

The increase in plasma chitotriosidase in GD

patients

is far morepronouncedthan theincreasein AP

activity,

which has

often been used as an

important diagnostic

hallmark of the

disease (16,

17).

Even in

asymptomatic

GC-deficient individ-uals a slight elevation in chitotriosidase

activity

was

_found,

whereas in these individuals APlevels were wellwithin the control range. Because ofthe

simplicity

and

sensitivity

of the assay, determination of chitotriosidase

activity

can

conve-nientlybe usedfor biochemical confirmation ofthe

diagnosis

of GD,inadditiontothedemonstration of

glucocerebrosidase

deficiency.

Surprisingly,

intwoofthe32 GD

patients

studied werepeatedly foundanalmostcompleteabsence

ofchitotriosi-dase

activity.

Inthesecasesresidualenzyme

activity

wasfound

tobe duetolysozyme, whichalsohas

catalytic activity

towards

5001 400 > 300 U .r

_{'._}

,, 200 E . N c E ₁₀₀ 0 c 0 0 A A A A AAA& A A A A _A A A, A A A AAA A 10 20 days of culture 30

Figure 2. Secretion ofchitotriosidase (filled triangles)andAP(open

triangles)inmedium of cultured monocytesduringdifferentiation into macrophages. Enzyme activities (means of a duplicate

experi-ment)areexpressed in nmol/ml medium per h. Valueswere corrected for thedailychange in medium.

the 4-methylumbelliferyl substrate, although clearly distinct from chitotriosidase.One ofthe parents ofa

chitotriosidase-de-ficientGD patientalso lacked enzyme activity, suggesting a

familial nature ofthe deficiency. Absence of chitotriosidase activitywasalso observed in 3of 50controlsstudied.

The question arisesas to what the biological function of

chitotriosidase is. Sincethe twoGD patients lacking chitotrio-sidaseactivity manifested characteristicclinical symptoms, it is

unlikely that chitotriosidase itself contributes to the clinical

presentation ofGD. On the other hand, the similarity between

lysozymeandchitotriosidase with respect to catalytic activity

40000 A

20004j.

30000

._2:

aD 20000 C _ C, -.2 E *° E_C 10000 0 -0 10 20 30 time(wk) 40 50

towards the same substrate suggests that chitotriosidasemay also haveafunction inhostdefense mechanisms,e.g., through cleavageofbacterial cell wall polysaccharide.

Anotherimportantquestion relates to the source of

chito-triosidase. GDis characterized by the presenceoflarge num-bersof macrophagesloadedwithglucosylceramide.Ittherefore

seemedlikelythat these so-called "Gaucher cells" are themain

sourceoftheplasmachitotriosidase activity.Our invitrodata supportthe hypothesis that the enzymeismacrophage derived. However, the rapiddecline in chitotriosidase levels afterthe startofenzymesupplementation therapy, preceding objective clinical improvement, suggests that the production of chito-triosidase isnot asimple function ofthe numberof lipid-laden

macrophages. This is also suggested by the poorcorrelation

between chitotriosidaselevels andSSI, which mainly focusses

on theextentoforganinvolvementinGD.Itismorelikely that

in GD aparticular state of activation ordifferentiation of mac-rophages oroftheir precursors leads to the excessive

produc-tion of chitotriosidase. The rapiddecrease in chitotriosidase activity during supplementation with GC may (initially)

re-flect an alteration in this state rather than a decrease in Gaucher cellmass. Thechangesinchitotriosidaselevels upon enzymesupplementationtherapy suggest that the enzymecan

beapromisingparameterforassessingtheclinicalresponseto treatment.Moreextendedstudiesareneededtorelateclinical

improvement to decrease in chitotriosidase levels.

Since activatedmacrophages contributetothe

pathophysi-ologyofmanydiseases, chitotriosidase activitywasdetermined

in plasma samplesofanumberofpatients with granulomatous immunological disorders (Wegener's granulomatosis, sarcoi-dosis) or granulomatous infectious diseases

(tuberculosis,

leishmaniasis,andleprosy).Elevated enzyme levelswereonly found for some patients with leishmaniasis and sarcoidosis

B

10 20 30 40 50

time(wk)

D

Figure 3. Decreaseinplasma chito-triosidaseactivityintime in four type 1 GDpatients (A-D)treated 10 20 30 40 50 with alglucerase (50 U/kg per mo).

(Hollak, C. E. M., and J. M. F. G. Aerts, manuscript in prepara-tion). However, in thesecases eventhehighest chitotriosidase levels did not exceed 1,800 nmol/ml per h, being clearly less than the lowest chitotriosidase activities found for symptom-atic GDpatients. In plasma samples of patients with acute and chronic myeloid leukemia (diseases that are commonly listed in thedifferential diagnosis of GD) chitotriosidase activities were within or slightly above the normal range.

It is of importance to note that chitotriosidase levels were found to be only moderately increased in the asymptomatic patients studied and > 100-fold in the mildly affected patients. This difference is unique with respect to secondary markers andsuggests that increasing chitotriosidase levels can prove to be asensitive harbinger ofclinical symptoms and consequently maycontribute to considerations concerning early therapeutic intervention.

Acknowledgments

Wegratefullyacknowledge the helpofDrs. C. Alberts and E. E. Zijlstra in collecting some ofthe plasma samples. We also thank Drs. R. GoudsmitandJ.M.Tagerfor their useful suggestions duringthe prepa-ration of themanuscript.Finally,weacknowledge the cooperation of the Gaucherdisease patientsandtheir families in The Netherlands.

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