P c d i a t . R c s . 11: 158-163 ( 1 9 7 7 )
C h r o n i c g r a n u l o n i a t o u s tliscnsc (CGII) glucosc-(1-pliosphatc t l c l i y d r o g c n n s e (G-6-PD)
leukocyte n i t r o b l u c t c trazoliilm
Multiple Leukocyte Abnor~nalitiesin Chronic Granulolnatous Disease: A Familial Stucly
\vith G-h-PI) lahility (0) o r elcficiency ( I ) in males, \\it11 glut;itIiio n e pcrosirlasc cleficiency in fem;alcs ( I S) ancl males (24). \vith !\ v:rriety of leiikocytc clizyllie activities Irere stucliecl ill all I l di~ilinishccl NAI>H osiclase ( 2 ) ancl NAIII'H osicliasc activity year-olcl fc111;1lew i t l ~cl~roiiicgrariulo~iiatousclise:~sc ( C G I ) ) alicl (10). anel ~vitli rlecrc;~scclsuperosiclc production (13). 111 the I : I ~ ~ report. ;I fiamily is stucliccl in \\liicli several ~ n c l n h c r s several ~ ~ ~ c i i i l )of e r hs e r L~iiiily.I.ei~kocyte ~ I I I c ~ s ~ - ~ - ~ ) ~ I o s ~ ) ~current clel~ytlroge~iasc (G-6-1'1)) activity was 1 7 I ~ I I I O ~ / I I protei~i ~ ~ I ~ / I ~m;~nifest I~ recurrent infections anel :allergic clisc;~sc,;~nrl\vho clisill tlie patielit; t\vo I)rotl~erswit11 s y l ~ ~ l ) t o lof ~ irecurrelit s 1)acteri:il pl~ay;abnormal h;actcriciilal function, impaireel rccluction of NI3'I' il~fectiolishave G-6-1'1) activities of 5 8 aiicl 3 7 ~ i ~ i i o l / ~ ~ i i i i / i clyc. i i g ancl :I variety of leukocytic enzyme clct;.cts. proteili; tlic acti,itcs of this c ~ i z y ~ i ill i c 1)otli parerits, 111;11er11:11 gr:~~icl~~iotlier, :111tl o11e :~clcIitio~~:il l)rotl~erwere witl~ili11orl11:11 lil~iifs. Sfor:~ge at 4" o r Iicatil~g;11 37" over :i 1 2 0 - ~ i i i ~period i revcalecl a lnarkecl lal~ilityof G-(,-PI) activity in tlie paticlit's K L is an I I-year-olcl C a ~ l c ; ~ s i ;fcmale ~n rcfcrrccl to Gcorgccells \rliicli coultl i ~ o I)e l stal~ilizecl11y tlic ;iclclitio~iof NAI)l' ;111cl 2-1iiercal)toeth:i1iol; this I;~l~ilitywas not see11 ill o t l ~ e rL1111ily t o \ w University I lospital for evaluation of chronic recurrent skin ~ ~ i c ~ ~ i ltestccl. ) e r s I\cti\itics of leukocyte gl~rtathiolicretluct:~se ancl respiratory infections \vliicll hcg;111 ; ~ t0 months of age. were rcclucecl in I ~ o t ~):~rerits l~ :~iicltlie ttro :iffectecl 111ale sil~liligs Within tlie first 2 ycars of life, a scrics of rccurrcnt tliro;lt infectiolis occurrcel \\liich ~ c r ctrcatccl successfully wit11 antibrill1 v ; ~ l ~ ~ofe 18, s 23. 23, alitl 2 4 ~ i ~ i i o l / ~ ~ i proteili, i ~ i / ~ ~ irespecg biotics. At 0 months of age, p l i c ~ ~ m o noccurrctl i;~ \\hiell clicl 11ot tively. Activities of leukocyte glutathione peroxitlase were re. patie~lt clcvelopccl ruhcola IS clucetl ill all of flie i~r~i~iecli:~le killlily 111e111l)erstestecl, nit11 v;~lues rccluire h o s p i t a l i r a t i o ~ ~7flic nlonths of age c o n c ~ ~ r r c n t l y \\,it11 her siblings. Shortly thcrc:aftcr. r:111gi11gfro111 I I .2 t o 4 3 ~ i ~ ~ i o l / ~ i ~1)roteili; i ~ i / ~ i tlie i g :~ctivityof tliis e l ~ z ~ lill~ tlic i c p:~tier~t stas 38.5. I , e ~ ~ k o c N ~ tr c\ l ) P col~teritin the she colltr:~ctccl varicclla ancl the pox lesions bccamc Iic:~vily h These were especially promi~ ' ; ~ t i c L~tlicr, ~ ~ t , :ilicl two :ifTectecl 111:1le sil~liligs\ \ e r e 16.5, 23.4, seconclarily infecteel ~ i t hactcri;~. nent o n the skin of the cyclicls anel perineum. Although tlc22.2. :ilicl 28.2 111iio1/151iii11/107 Ieiikoc~tes,respectively. crc:~sccl in frccluency. the recurrent throat ilifections have continueel to the present, ancl over the past 3 years. recurrent skin infcctions have also :~ppe;~recl. 'l'hese have consisted priChronic g ' : ~ ~ ~ i ~ l o ~ i i : ~tlisc:~se t o i ~ s rrl)reserits a Iietcroge~~oiis col- marily of vcsiculopi~stular lesions localized to the skin of the Icctioli of Iei~kocytee~izy~~i:ific a l ) ~ i o r ~ ~ ~ a l ~rhicli i t i e s result ill a perioral regions ancl Icgs. l \ v o ycars ago, the p;~ticntdeveloped cliliic:~l picture of r c c ~ ~ r r e l I~:~rtcri:~l it ilifectioli. 'l'hc fi11:11 co111- pharyngitis and a n ~tbscesscclcervical lymph noclc which rciiiori ~):~fl~\r:iy ill flie ~)aflioge~iesis of the clisorcler clerives fro111 cluireel surgical drainage. After a 10-cl:~y course of parentcral penicillin therapy, she \v:~splacccl o n tl:~ily prophylactic osacillin clefeclivc II,O, procluctioli o ~ r i i i gto a v:iriety of e1izy111:itic :I!)~ ~ o r r ~ i : ~ l i\ritliiri t i c s o r lilikccl t o flit Iiesose ~~ioriopliospliatc sl~ulit f o i approximately 1 year uliich significa~itlylesscnccl the frc(IIRll'). 'l'lic stuclies of f l ~ ekiliclrccl ill t l ~ epreselit rel)ort lcl~cl clucncy and severity of the rccurrcnt skin infcctions. furtlier s111)port for tlic celitr;~lrole of Nr\I)I' ;IS a co~itrolli~ig T h e p:aticnt was ;a procluct of a full tern1 normal pregnancy. Imrnuriiz:rtio~ls.i~icli~rli~ig znialll~osvaccin:ition. wcrc pcrforlncd ~iiecli:~~iisii~ of iior111:11 l e ~ ~ k o c y l):~ctericicl:~l te f1111ctio11. \vithout complici~tion.'I'\vo \veeks after rubella immunizatio~l. lio\vcver, rash. rnal;~iseancl arthralgi;~occurrccl. \vhich suhsicled 'l'lie clinic;al entity. C'GI). is ;a cli\orclcr of ncutropliil function \vithin 3-5 clays. She has hael collt;~ctclermatitis clue t o poison ivy in ~ h i c l itllc Icukocytcs of affectctl patients can ph;~gocytosc i11ic1 poison o a k . hactcria normally l1ut have i~npaircclintraccllul;ar killing of ccrI'liysiical cs;amin:ation rcvcalecl ;an ;alert. coopcr;~tivc.well tlct;ain hactcri:~ ( 2 0 . 2 0 ) . hletabolically. Icukocytcs of affecteel velolxcl I I-year-ole1 Caucasi;~n fcmale ~ v i t hfair skin ancl reel paticlits fail to slio\v tlic riorlnal s t i ~ n ~ ~ l a t of i o nrespiratory activ- liirir. Scvcr:al scars were present on tlie skin at the sites of ity. incrcasccl 0, consl~lnptioll.osielation of glucosc through the previous skin infections ancl surgic;al proccclurcs. T h e spleen tip llhll'. anel generation of I1yclrogen perositlc (11,O2). events was p;~lp;ablc 1-7 em hclo\v tlic left cost:~lmargin. A graclc I-II/ associatctl \villi norliial b;~ctericicl;~l ;ictio~i( 3 . 17). 7'licrc is a VI innocent systolic Illrlrmilr \\as hcartl along the left sternal concomitant failure of these affcctccl cells to change tlie osiclizccl borcler. colorless form of nitroblue tctrarolium (NI3.I') clye to its I>luc Complete hlooel count revc:~lccl a hcrn~atocritof 3 7 % ;111cl a rccl~lcccl counterpi~rt cl~rillg pli;~gocytosis. provicli~ig ;I i~seful total Icukocyte count of 7,500 cells/mm:' \vith 3 0 % poly~iiorphocliagnostic test for the tliscasc ( 4 , 7 5 . 2 7 ) . nuclear leukocytes, 0 4 % lymphocytes, 7 % Imonocytcs, i111tI 4 % Originally described in males :IS a synclrome with a n X-linked cosinopliils; cosinopliilii~\v;~salso noteel o n scveri~lsuhsccluent mode of tr;ansrnission ( 9 , 10. 34). C G D lias been clcscrihcd in csaminiations. Urinalysis was \vithin normal limits. Protein elccmale aricl fcmalc patients \vitli a n ~ ~ u t o s o m arecessive l nlodc of trophorcsis, i1iimunoclcctro~>I1orcsis.:~ncl chcmotactic studies inhcritarice (18, 24. 2 8 ) . A myriacl of Icukocyte enzyme clcfi- (pcrformccl hy D r . Pctcr A . Warcl) ivcre all norm;~l.A hlono cicncics have hccn clcscrihccl. 'l'lie entity lias bccn :~ssociatccl spot test (Ortho) and fchrilc agglutirlins were negative. Stools ~
~
159
LEUKOCYTE ENZYhlATIC ABNOllhiALlTlES were negative for ova and p:lrasitcs. and blood irrc;i nitrogen, crentininc. blood sugar, alkaline phosphutnsc, scruni glutnmicoxaloacetic a n d serum glutamic-pyruvic transaniinases, a n d lactate clcliytlrogeniisc were all within normal limits. A t the time of referral. S ~ r r p l ~ ~ ~ l o c oc~pit1crttritli.s. c~(~r~.r Strc~l~rococcrts ~ ~ i r i d ~ t;iricI rs. g r o u p 1) P-hemolytic streptococcus were recovered from infected skin lesions. .l'ests of clclaycd skin hypersensitivity to m u m p s virus \\.ere positive hut negative t o tuhcrculin. cocciclioitiin. blastomycin. and 1iistopl:ismin.
NI:UI'ROPIIIL ENZYhlE
h1ATERIALS AND hlE'I't1OIlS SUBJECTS A n 1 I-year-old fernale and her family \\,ere stutlictl ;it Gcorgetown University IIospital (Table 1). Age-adjusted and adult subjects were inclutled a s control suhjects for the biochemical ant1 bactcricirlal studies clcscrihccl below. PRI~l'Allr\TION 01: LEUKOCYTE SUSPENSION hletliocls for the preparation of a relatively rich suspension of granirlocytes \\.ere those clcscribcd by Baehncr ; ~ n t lNathan (4) wit11 minor rnodific:ition. as previously clcscribcd ( 6 ) . Differential count of this lluitl revealeel values consistently i l l excess of 9 0 % polymorplionuclear leukocytes.
T h e clumititativc NL3.f tcst was performed by the method of Baehncr and Nathan (4). results being cxprcssccl as the cliango in optical density pli;~gocytosingvalues for 7 . 5 x 10" cells for 15 min at 37". Bi\C'I'I:RICIDAL
DE~I'ERhllNAl'IONS
Cell homogcnatcs were preparecl fro111 frozen cruclc cell suspensions by methocls descrihccl previously ( 6 ) . T h e sirpcrn;it;ints were analyzed for protein content (33) ancl the enzymatic assays were pcrformcd a s clescrihcd below. - 1 - 1 ~ ,ict~vitics .~ . ' of G-0-PD. 0-pIiospliogluc~)~iiitc tlchydrogcnasc (6-PGD), and NADP-specific glutathione reductase in white cell lysates \\,ere tlctcrmincd by the rnctliocl of Richterich ( 3 1 ) . Glutathione peroxitlase activity was determined by the method of Paglia a n d Valentine (26). T h e results of all leukocyte enzyme activities a r c reported a s nanornoles per min per rng protein a t 75". Leukocyte N A D P content was assayed according to the procetlure of Klingcnbcrg ( 2 2 ) and is rcportcd a s nnnomolcs per 15 min per 10' leukocytes. T h e optical densities were clctcrmined with a spectrophotomctcr using I-cm cuvcttes that were maintained at 25' by means of a circul;~tirigwater b a t h . G-&PI) LABILITY
A N D hllXlNG
EXPElllhlEN'I'S
Experiments were performed t o determine the effect of stabilizing agents o n G-6-PD activity during scri:~l incubation periods a t 37" accorcling t o rncthotls prcviously described (6). Leukocyte homogcnates, prep~lrcclfrom leukocytes frozen a t -70°, were tcstccl both in the absence ancl prcscncc of 6 pM N A D P plus 2 5 m h l 2-mcrcaptoethanol (2-hlE) a t 0, 3 0 , 6 0 , a n d 120 min of incubation at 37" (Tiible 3 , Fig. 2 ) . I n o r d e r to assess the possibility of a G-6-PD enzyme inhibitor, leukocyte homogenates from the patient and a normal control subject were rnixcd in equal proportions and lability studies were performed in accordance wit11 the methods just describccl. T h e results \\..ere compared with the lability stuclics of the patient's and the control's cells when tested individually (Fig. 3 ) .
TEST
CLINICAL DATA T h e hiictcriciclal assay was pcrformcd by the mcthocl of Quie (11. ( 7 9 ) using S~rr~~lr!~lococcr~.s (rrlr(,rl.s :IS a test strain. T h e Cli~iicaldata o n thc patient's family arc give11 in Table 1 a n d results were cxpressecl :IS the pcrccnt:ige of viable bacteria re- :~ccorclingt o the pctligree slio\vn in Figure 1 . It can be seen that, maining after 170 riiin of i ~ i c ~ r l x l t i cwith ~ ~ i p o I y r i i ~ ~ r p l i o ~ i ~ ~ c of I c :the ~ r 17 n i e ~ n h c r sfor \vliom information was available, infecleukocytes. tions were seen ill the maternal grc;itgranclrnothcr (11-2). the
c.1
Falllily mcrnhcr GG A1 GGF KA KC
No. in pc~ligrcc I- I I-? 11-1 11-7
Age. Ilclatioli to prohand -hlatcrlial grcatgr;inclinothcr hlatern;il grcatgr:illdfathcr hlaterlial grandfather hlatcrn;~lgranclmothcr hl;~tertial\tcpgr;illilf:ither Uncle by marriage hlutern;il aunt hlother
WI* C'C'
h1C
IV- l IV-2 1V-3
First cousin First cousin Brother
Scs F hl hl
Died 71 Ilicd 8 2 54
F
54
-
Age at onset of infcctions. years Unknown 0 0 44
Clinical symptornatology Skin infections. chronic bronchitis tfe;ilthy Healthy Cystitis. rash. rcpcatc~lb;~cterinl upper rcspiratory infections l lcnlthy Rlicumntic fever, hc;ilthy Chronic sinusitis, multiple allergies Eczcina, recurrent periodontal and urin;iry tract infcctions Allcrgics since chiltlh(~od Ilepcated pliary~~gotolisillitis, p;uicytopc~iia Ilcpcatcd bacterial pneumoni;~~ cliagriosed as liypogammaglob~~lincliiia ;ind treated with y-glohulin, age 7-6 years hlilk ;rllergy, sillusitis hlilk allergy, sinusitis. hordcolum h n~o-1 year Asthnl;~,recurrent pliaryr~goto~isillitis;ind otitis mcditi, urili:iry tract infcctions. boils 011 skin. granuloma removeel i i i 1967 from arm Ilcpeatccl pliaryngotol~sillitis;incl otitis rnc~li;i(see text) Atopic cczcnl;i, milk allergy, no recurrent infcc-
niothcr (111-3). ancl t \ \ o siblings (1V-3 ancl IV-4). A strong patielit ;~nclthe t u o ni;rlc silllings \vith impairetl NB'I' clye rccluchistory o f allergic clisc;~sc\ \ ; I S obtainccl in scvcr;ll family mcm- tion Iiacl significantly rcducecl G-0-I'll ;~ctivity\vlicn tcstccl i l l the I x r s (11-7, 111-7. 111-3. 111-4. IV- I , IV-2. IV-3. IV-0). A l t l i o ~ ~ g l i absence of N A I I P ant1 7-RIE at 37' (Tahlc 3 ) . .l'lic r e s ~ ~ l ot sf :I chilrlhoo~lhistory of liypogammagIoI>11li11c1iii:1 was oht:~inctlin Inore estcnsivc ilivcstig:~tion of G-0-1'1) Iahility o n the paticnt ancl the immccli:~te fc1mily itre slionn in Figi~rc3 . All extreme ;I matcrlial uncle (111-0). tests of this incliviclual's serum imniunolability o f Icukocytc G-0-PI> \vas observccl in the p ~ ~ t i c ncvcn t. globulin Icvcls tvcrc founcl to be \vitliin ~iorniallimits. v,Iicn tcstccl in the presence of 7-hIE anel N A I I P . G-6-1'1) lallility was not clctcctcd in any other farnilv members \\hen N13.r AN11 l3AC1'~:ls1
G-6-PD + NADP + 2-ME
GSSG rcduclase
6-PGD
GSH pcroxidase
Proband Father Mothcr Brother Brother Brother Maternal grandmother Maternal aunt 118.0 + 4.0 129.8 + 12.5 46.2 + 2.1 30.9 + 0.5 82.7 2 3.23 01= 37) (11 = 4) (n = 44) (11 = 26) (11 = 26) Enzyme activity values expressed as nanomoles per min per mg protein 5 SE mean. G-6-PD: glucose-6-phosphate dchydrogcnase; 2-ME: 2mcrcaptocthiinol; 6-PGD: 6-phosphogluconate dchydrogenase; GSSG: oxidized glutathionc; GSH: reduced glutathionc. No enzyme stabilizing agents added. 3tatistical significance compared to normal (Student's I-test); P < 0.005. P < 0.01. P < 0.025. Control
n
':v-
80
47
- -.--
K.C.
-:- -- _- - .
-1t -A*
\
Glycogen
B.L., M.
'S.E.
H202 (Bocleriol)
Glucose-Glucose-6-P
\control -- - - .------' N.L. ---4
Bactericidal Complex
W.L. 6-R~bulose j-Phosphate (Leukocylic)
a Glutathione peroxidase Loclale-Pyruvate
@
x'
K.L.
-
I
1
K r e b s Cycle
"0
90 120 Incubation Time (Minutes at 37OC) 30
60
Q N A D P H S p e c i f ~ cglutothione reductase Q Glucose-6-phosphate dehydrogenase @ N A D P H oxidase Q) 6-Phosphoglucanole dehydrogenase
@ N A D H oxidose
0 Phosphoglyceraldehyps
dehydrogenase
@ NADH-linked loclic dehydrogenase @ Catalose MPO=myeloperoaidose
Fig. 2. Leukocyte glucose-6-phosphate dchydrogcnase lability of patient ( K L) anrl her inimcdiatc family.
Control Mixing Experiment (Control t Patient)
K.L. (Patient)
s
OO
30 60 90 120 Incubation Time (Minutes at 3 7 O ~ ) Fig. 3. Leukocyte glucose-6-phosphate dchydrogcnase mixing cxperimcnt. glutathione peroxidase was first described in females with C G D (18), and subsequently in a male child with this syndrome (24). In contrast to the present studies, however, a lability of G-6-PD was not detected. Another two patients with C G D have been shown to have deficient leukocyte superoxide production (13). The iniportance of the superoxide radical is that it can be bactericidal itself, o r it may be further reduced to H,O,. The H,O, formed may react with additional ~noleculesof superoxide to
Fig. 4. Leukocyte cnzymc biochcniistry. form hydroxyl radicals o r nlay participate in the production of bactericidal aldehydes (32). A n iodination defect has also been described in the leukocytes of patients with C G D (21). I n the present studies the labile G-6-PD enzyme in the leukocytes of the two male siblings could be stabilized by the addition of N A D P and 2-ME, indicating the importance of this cofactor and of sulfhydral linkages in stabilization. The patient's G-6-PD was so severly labile, however, that stabilization was not possible even with the use of both of these agents (Fig. 1). The leukocyte G-6-PD lability in this family could be explained by the reduced content of leukocyte NADP, a deficiency of stabilizing factors, o r an altered N A D P to NADPI-I ratio as suggested by the studies of Erickson et a l . (15). This is consistent with the findings of Yoshida (35), which indicate that monomeric G-6-PD is linked together by a ~noleculcof NADP, forming an active dimer. Abnormalities in the glutathione system were also detected in this kindred (Fig. 1, Table 3). The finding of diminished activity of glutathione peroxidase is of interest because of the data of Strauss et a l . (33). These authors have suggested a primary role for the glutathione system in initiating the biochemical cascade resulting in the marked increase in HMP activity and H,O, production necessary for normal bactericidal activity. The deficiency in NADPH-specific glutathione reductase detected in the two male siblings with G-6-PD lability and in both parents further differentiates this particular syndrome of neutrophil dysfunction from the classic X-linked form in which an elevated activity of leukocyte glutathione reductase was reported (18).
In the franlc\vork of the schcm:~proposetl by KIcb:rnoff (90). 2. tlcpicting the inter-rclationsliips of the Icukocyte enzymes (17ig. 4). thrcc recent cornmunic;rtio~~s help to elucitlatc the pirthogcnctic nlcch:rnisrns involvctl in this kindred's tlisortlcr. 3. O n the lxrsis of results obtained using a grir~iulocytc-frcc 4. system in \\hich s:rnthinc oxitlasc is suhstitutccl for NAIIPfI oxitlase. Curnuttc (11. (1 1) speculated that the hulk of the 5. oxitlation that occurs \\hen NALII'FI. g r a n u l o q t c particles. ant1 m;rng:rnese ;ire illcuhatctl together takes plircc via :I noncnzy0. nlatic free r:rdical chain re;rction. 'fhcy concludecl that the function of NA1)I'Il oxitlasc \voultl he t o generate the supcrositlc rirtlical necessary t o initiate this reaction. Thcsc results arc pcrti7. ncnt in that these iruthors h;rve previously reported (1 7 ) a tlcfcct in the pyritlinc nuclcotitlc tlepcndcnt protluctioli of supcrosicle in the leukocytes of three p;rticnts n i t h X-linked C G I I . 'l'hey 8. favorctl NAIlI'I1 ositl;rsc as the tlcfcctivc cnzymc in thcsc patients. Ilohn antl Lehrer (16) have dcrnorlstrirtcd that the activ0. ity of NADPFI oxidasc in polynlorphonuclcnr Icukocytcs from patients with C G I I was 8% of normal. These authors conclutlecl 10. that the absence of a cyanide-insensitive N A D P H oxidasc o r a deficiency in its activntioli is the primary metabolic defect in the X-linked C G D syntlrome. O u r data is consistelit with both I I . of thcsc fillclings in that a tlcficiency in the N A D P H oxitlase system could result in reduced leukocyte N A D P content and the 12. G-6-PD lability. Since N A D P is a regulirtor of H h l P activity (5). its unavailability woultl inhihit the direct oxitlation of glucose through the HhlI' cithcr directly, o r secondarily by il1activ;rtion 13. of G-6-PD, accounting for the well known tlecreasctl H h l P activity in C G D leukocytes (3, 17). It is tempting to spcculirte that ;I dcfcct in the N A I I P generating system could :rlso be related 14. t o the tlccrcasetl activity of glutatliionc pcroxidasc, since this en- 15. zyme requires H,0, irs a substrrrte (Fig. 4). Thus, a clefcct in the NAIIPFI oxidasc system coulcl not only account for our biochemical findings, but also the inability of the leukocytes to I h . protlucc lI,O, supcroxitle, thereby causirlg a tlefect in their bac- 17. tericidal function. Although the :rffcctccl m;rlc mcmbcrs of this family have not hat1 fulrninant bilctcrial infectiolls. \vc feel that their clinical 18. manifestations arc compatible \vith the courses of patients prcviously tlcscribctl (8, 9 5 ) \vho h;rvc m:rllifestcd o rniltler clinicirl 1 0 . form of C G D . 'I'hc heterozygosity dcmonstr;rtctl in this kintlrcd is consistent with a n :rutosomal rcccssivc mode of tr;rnsrnission. 2 0 . 'l'hc patient. \vho manifests the most scvcrc clinical illness. 2 1 . u.oultl he l~omozygous;the unaffected male sibling. ~ \ l l I ant1 . the pirrcnts. NL ancl IVL. woultl he unaffcctctl carriers of the gcnc; ant1 the other two male siblings. 1)L and Ill,. \vo~lldbe hetcrozy- 22. gotcs with an intcrrnctliatc form of expression accounting for both their milt1 clinic;rl fi~icli~igs. their impairctl N B T tlyc rcduc- 2 3 . tion, ancl tlccrcascd Ixrctcricidal activity. T h e st~rtly of this kinclrcd has provirlcrl further cvitlcnce tliirt CGI) represents a 24. s11cctrt1111of Icukocytc enzyme itbnormalitics anti h:rs i~nplic;rtctl G-6-1'1) lability ;rntl the NAIII' gcrlcrating system :IS a n etiologic 25. firctor i l l the ncutropllil tlysfunction of this kintlrctl.
cytic C'cll in llost K c s ~ \ t a n c e lip. , 173-200 ( R ; ~ v c nPress. New Y o r k , 1075). Baehncr. R . L.. a n d Karnovsky, h l . L.: Deficiency of reduced nicotin;~mideadenine ilinuclcotidc oxidase in chronic gr;~nulornatouscliscase. Science, 162: 1 2 7 7 (1968). 13;lehncr. Ipsosim;ttcly 0 3 2 mOsm/liter. 13iliruhin-human alhurllirl solutions \ \ e r e prepared by clissolving I>iliruliin (Sigma) in 0 . 5 N N a O l l and adding the I>ilirullin t o a l l > u ~ i i i ~(crys~;tllinc, l C'11ttc.r) tli\\ol\rCd i l l 0 . 0 5 5 X I phosphate buffer, p1~17 . 4 , ionic strength 0 . 1 5 . All I>iliruliin solutions were l \ ~ r ~ acids i ~ ~ oill so111tio11(lo 11o1irl)1)c;ir t o i ~ ~ t e r f c vit11 r c ljiliru- p r c p ; ~ c c lanel maintai~icclin the cl;lrk o r sul~cluccllight. 1:in:tl p t 1 I I ~ Itr;~lrsport. I I';~re~iter:~l : r I i ~ ~ i c ~ ~ t : iwit11 t i o ~ ia r ~ ~ i r :rcicls lo S I I ~ I I I I I of bilirubin-all>l~mins o l u t i o ~ l swas 7 . 4 . I)c :I safe proccclurc ill j:~rrlrclicecl p:ltic~rts. 'l'llc intcractio~lof ;\mino a c i ~ l swith hilirul>in-all,unlin cornplcacs : r ~ l c l tr;~nsport\vcrc stueliccl by cotllpctitivc hincling :rssa!,s using c l i o l c s ~ r a n l i n e;trlcl Sepliaclcs gel filtration. the perosiclasc I'arcntcral fcccling is frcclucntly rcclr~il.c~l in prcm;tturc ancl assay. red cell upt:~hcof raclioactivc Ililirullin, ancl lly a n arialysis sick nc\\ horn infants \\.ho a r c ~ln;tl>lcto take a ~ l c c l t ~ a ~t el u t r i t i o n of :rbsorptio~l spcctr;r. I>y ~ n o u t h . I'hc p:~st tl~,c:~clch : ~ \ I11.ottglit tcc.litiic;~l ;icIv:~nccs \\ hich rnahc i t possible t o supply not only c;trl)ohy~lratc.I n i t ; ~ l \ o fat ;tnd a m i n o ;rciels by ;III in1r;tvcnou.; route. Uy these means. suslainctl gso\\th has h c c n ; ~ c l i i c v e ~inl 170th prcm:tturc ,inel term C'o~llpctitivc bincli~lg I ~ c t \ \ , c c ~a l l ~ u m i n a~lcl cholcstyramiric ( 1 2 ) \\as studiccl 11sir1ga ~ ~ r c i c c t l u rnloclil'ic~l e from Sch~lliclor ol. i1lf:111ts(2. 3 ) . 1Jnconji1g;ttctI I~ypcrl>iliruhincrni;~ i \ frcclucntly p s c s c ~ ~int sick ( 1 0 ) . I3ilirul>in-:tll~111iii11solutions \ \ e r e prcp;rrccl ,o cont;rin 5 gl I 0 0 1111 1111111;111sc1.11111 ;1lhurlli11;I ~ i l i r ~ r l ~ i ~ ~ / ; t l 111oI:rr l ~ ~ ~ ~ i ir;rtios r~ of 11c\vl>ornsrcccivi~lghigll c o n c c ~ l t r a l i o n sof a ~ n i n o;~citlsancl/or 0.5, 1 .O. 2 . 0 ; anel ;111lino;leiel c o r l c c ~ l t s ; ~ t i o f~ 0~ s. 0.SO. ;~ncl2 . 6 7 lipids. Silicc tllc possil7ility of kcrnictcrr~sis kn0\\11 t o he ing/lOOnll. /-\I) cclual volunlc of p r c \ \ ; ~ ~ l ~ c hc ol l e s ~ y r a ~ n i ~( 1l c2 ) crc;tsccl Iiy tllerapcutic agents \\ hich intcrl'crc \\it11 Ilinclirlg of c stirre11 c o ~ ~ \ t : l ~ ~ t i y . \ \ a s acl~lccl;111cl tllc ~ n i ~ t u s\v;rs bilirulii~lt o pla\nla ; ~ l l ) u ~ n (7. i n 1 I ). i t is inrportant t o c v a l u : ~ t c suspc.~~\irin /\licluol\ \ \ e r e rctno\.c.cl ;I{ 0 , 1 5 . 3 0 . 0 0 , ;11111 0 0 l l i i ~ lof C Y P O S ~ I ~ ~ o n hilisl~hir~ transport. tile cfl'cct of p ; ~ r c n t c r ; ~n li t t ~ . i c ~scilutio~is it ;11ic1I'illcr~cifree of 1.c5irr t l i s o ~ ~ g~h1 ; 1 \\\oo1. s ' 1 ' 1 1 ~e ~ ~ ~ e c ~ i l r ; t t i ~ ~ i 'l'llis stucly e ~ , a l ~ ~ atile t c s i~lflucnceof ;r c o ~ ~ l r n e r c i a l l;tv:ril;thlc y ;~l \v:I> c l c ~ c ~ - ~ ~ by ~i~iccl ~ L , I ~ I : Iit1 ~ Ii ~I i~c lIi I\ i~~ I ~ ti'iltr:~tcs lli~i t o 1111111:111 of l~iIir-t~l)i~i syntllclic :rmino acid soluliorl 0 1 1 I > i l i ~ - ~ ~hi~iclillg the ~nc,tllc>clof hlallo), ant1 Il\.cl!t~ (0). ('o~llrcilc s l > c r i ~ n c ~ iilll s scsuln ;1ll)1111li1i : I I ~ C I red h l o o ~ lcell\. 'l'llc effects of s y r ~ t l ~ e t arl~illo ic :~citls o n I)ilirrrl)i~itrarlsport \ \ e r e i l ~ ~ e s t i g a l c cnit11 l corlrl)ctiti\e I ) i ~ ~ c l iassays, ~ ~ g perosiclasc l i c recl cells, ii~rtl :Issay, isotol)ic sfuciics of I)ilirlrl)i~r~ ~ l ) t ; ~11.) clin'crence spectroscopy. licsults intlic:ltctl that a~rririo:lcitls llatl 110 sig~~ific:rl~t cl'fcct 011 tile c I i s t r i l ) ~ ~ t iof o ~l)ilir~rl)i~i i a t p i g ~ ~t ~ o c oll)urr~illrlrol:lr riltios liliclj t o I)c c~icourrtcretlill clirlical situatiorrs.
C h r o n i c g r a n u l o n i a t o u s tliscnsc (CGII) glucosc-(1-pliosphatc t l c l i y d r o g c n n s e (G-6-PD)
leukocyte n i t r o b l u c t c trazoliilm
Multiple Leukocyte Abnor~nalitiesin Chronic Granulolnatous Disease: A Familial Stucly
\vith G-h-PI) lahility (0) o r elcficiency ( I ) in males, \\it11 glut;itIiio n e pcrosirlasc cleficiency in fem;alcs ( I S) ancl males (24). \vith !\ v:rriety of leiikocytc clizyllie activities Irere stucliecl ill all I l di~ilinishccl NAI>H osiclase ( 2 ) ancl NAIII'H osicliasc activity year-olcl fc111;1lew i t l ~cl~roiiicgrariulo~iiatousclise:~sc ( C G I ) ) alicl (10). anel ~vitli rlecrc;~scclsuperosiclc production (13). 111 the I : I ~ ~ report. ;I fiamily is stucliccl in \\liicli several ~ n c l n h c r s several ~ ~ ~ c i i i l )of e r hs e r L~iiiily.I.ei~kocyte ~ I I I c ~ s ~ - ~ - ~ ) ~ I o s ~ ) ~current clel~ytlroge~iasc (G-6-1'1)) activity was 1 7 I ~ I I I O ~ / I I protei~i ~ ~ I ~ / I ~m;~nifest I~ recurrent infections anel :allergic clisc;~sc,;~nrl\vho clisill tlie patielit; t\vo I)rotl~erswit11 s y l ~ ~ l ) t o lof ~ irecurrelit s 1)acteri:il pl~ay;abnormal h;actcriciilal function, impaireel rccluction of NI3'I' il~fectiolishave G-6-1'1) activities of 5 8 aiicl 3 7 ~ i ~ i i o l / ~ ~ i i i i / i clyc. i i g ancl :I variety of leukocytic enzyme clct;.cts. proteili; tlic acti,itcs of this c ~ i z y ~ i ill i c 1)otli parerits, 111;11er11:11 gr:~~icl~~iotlier, :111tl o11e :~clcIitio~~:il l)rotl~erwere witl~ili11orl11:11 lil~iifs. Sfor:~ge at 4" o r Iicatil~g;11 37" over :i 1 2 0 - ~ i i i ~period i revcalecl a lnarkecl lal~ilityof G-(,-PI) activity in tlie paticlit's K L is an I I-year-olcl C a ~ l c ; ~ s i ;fcmale ~n rcfcrrccl to Gcorgccells \rliicli coultl i ~ o I)e l stal~ilizecl11y tlic ;iclclitio~iof NAI)l' ;111cl 2-1iiercal)toeth:i1iol; this I;~l~ilitywas not see11 ill o t l ~ e rL1111ily t o \ w University I lospital for evaluation of chronic recurrent skin ~ ~ i c ~ ~ i ltestccl. ) e r s I\cti\itics of leukocyte gl~rtathiolicretluct:~se ancl respiratory infections \vliicll hcg;111 ; ~ t0 months of age. were rcclucecl in I ~ o t ~):~rerits l~ :~iicltlie ttro :iffectecl 111ale sil~liligs Within tlie first 2 ycars of life, a scrics of rccurrcnt tliro;lt infectiolis occurrcel \\liich ~ c r ctrcatccl successfully wit11 antibrill1 v ; ~ l ~ ~ofe 18, s 23. 23, alitl 2 4 ~ i ~ i i o l / ~ ~ i proteili, i ~ i / ~ ~ irespecg biotics. At 0 months of age, p l i c ~ ~ m o noccurrctl i;~ \\hiell clicl 11ot tively. Activities of leukocyte glutathione peroxitlase were re. patie~lt clcvelopccl ruhcola IS clucetl ill all of flie i~r~i~iecli:~le killlily 111e111l)erstestecl, nit11 v;~lues rccluire h o s p i t a l i r a t i o ~ ~7flic nlonths of age c o n c ~ ~ r r c n t l y \\,it11 her siblings. Shortly thcrc:aftcr. r:111gi11gfro111 I I .2 t o 4 3 ~ i ~ ~ i o l / ~ i ~1)roteili; i ~ i / ~ i tlie i g :~ctivityof tliis e l ~ z ~ lill~ tlic i c p:~tier~t stas 38.5. I , e ~ ~ k o c N ~ tr c\ l ) P col~teritin the she colltr:~ctccl varicclla ancl the pox lesions bccamc Iic:~vily h These were especially promi~ ' ; ~ t i c L~tlicr, ~ ~ t , :ilicl two :ifTectecl 111:1le sil~liligs\ \ e r e 16.5, 23.4, seconclarily infecteel ~ i t hactcri;~. nent o n the skin of the cyclicls anel perineum. Although tlc22.2. :ilicl 28.2 111iio1/151iii11/107 Ieiikoc~tes,respectively. crc:~sccl in frccluency. the recurrent throat ilifections have continueel to the present, ancl over the past 3 years. recurrent skin infcctions have also :~ppe;~recl. 'l'hese have consisted priChronic g ' : ~ ~ ~ i ~ l o ~ i i : ~tlisc:~se t o i ~ s rrl)reserits a Iietcroge~~oiis col- marily of vcsiculopi~stular lesions localized to the skin of the Icctioli of Iei~kocytee~izy~~i:ific a l ) ~ i o r ~ ~ ~ a l ~rhicli i t i e s result ill a perioral regions ancl Icgs. l \ v o ycars ago, the p;~ticntdeveloped cliliic:~l picture of r c c ~ ~ r r e l I~:~rtcri:~l it ilifectioli. 'l'hc fi11:11 co111- pharyngitis and a n ~tbscesscclcervical lymph noclc which rciiiori ~):~fl~\r:iy ill flie ~)aflioge~iesis of the clisorcler clerives fro111 cluireel surgical drainage. After a 10-cl:~y course of parentcral penicillin therapy, she \v:~splacccl o n tl:~ily prophylactic osacillin clefeclivc II,O, procluctioli o ~ r i i i gto a v:iriety of e1izy111:itic :I!)~ ~ o r r ~ i : ~ l i\ritliiri t i c s o r lilikccl t o flit Iiesose ~~ioriopliospliatc sl~ulit f o i approximately 1 year uliich significa~itlylesscnccl the frc(IIRll'). 'l'lic stuclies of f l ~ ekiliclrccl ill t l ~ epreselit rel)ort lcl~cl clucncy and severity of the rccurrcnt skin infcctions. furtlier s111)port for tlic celitr;~lrole of Nr\I)I' ;IS a co~itrolli~ig T h e p:aticnt was ;a procluct of a full tern1 normal pregnancy. Imrnuriiz:rtio~ls.i~icli~rli~ig znialll~osvaccin:ition. wcrc pcrforlncd ~iiecli:~~iisii~ of iior111:11 l e ~ ~ k o c y l):~ctericicl:~l te f1111ctio11. \vithout complici~tion.'I'\vo \veeks after rubella immunizatio~l. lio\vcver, rash. rnal;~iseancl arthralgi;~occurrccl. \vhich suhsicled 'l'lie clinic;al entity. C'GI). is ;a cli\orclcr of ncutropliil function \vithin 3-5 clays. She has hael collt;~ctclermatitis clue t o poison ivy in ~ h i c l itllc Icukocytcs of affectctl patients can ph;~gocytosc i11ic1 poison o a k . hactcria normally l1ut have i~npaircclintraccllul;ar killing of ccrI'liysiical cs;amin:ation rcvcalecl ;an ;alert. coopcr;~tivc.well tlct;ain hactcri:~ ( 2 0 . 2 0 ) . hletabolically. Icukocytcs of affecteel velolxcl I I-year-ole1 Caucasi;~n fcmale ~ v i t hfair skin ancl reel paticlits fail to slio\v tlic riorlnal s t i ~ n ~ ~ l a t of i o nrespiratory activ- liirir. Scvcr:al scars were present on tlie skin at the sites of ity. incrcasccl 0, consl~lnptioll.osielation of glucosc through the previous skin infections ancl surgic;al proccclurcs. T h e spleen tip llhll'. anel generation of I1yclrogen perositlc (11,O2). events was p;~lp;ablc 1-7 em hclo\v tlic left cost:~lmargin. A graclc I-II/ associatctl \villi norliial b;~ctericicl;~l ;ictio~i( 3 . 17). 7'licrc is a VI innocent systolic Illrlrmilr \\as hcartl along the left sternal concomitant failure of these affcctccl cells to change tlie osiclizccl borcler. colorless form of nitroblue tctrarolium (NI3.I') clye to its I>luc Complete hlooel count revc:~lccl a hcrn~atocritof 3 7 % ;111cl a rccl~lcccl counterpi~rt cl~rillg pli;~gocytosis. provicli~ig ;I i~seful total Icukocyte count of 7,500 cells/mm:' \vith 3 0 % poly~iiorphocliagnostic test for the tliscasc ( 4 , 7 5 . 2 7 ) . nuclear leukocytes, 0 4 % lymphocytes, 7 % Imonocytcs, i111tI 4 % Originally described in males :IS a synclrome with a n X-linked cosinopliils; cosinopliilii~\v;~salso noteel o n scveri~lsuhsccluent mode of tr;ansrnission ( 9 , 10. 34). C G D lias been clcscrihcd in csaminiations. Urinalysis was \vithin normal limits. Protein elccmale aricl fcmalc patients \vitli a n ~ ~ u t o s o m arecessive l nlodc of trophorcsis, i1iimunoclcctro~>I1orcsis.:~ncl chcmotactic studies inhcritarice (18, 24. 2 8 ) . A myriacl of Icukocyte enzyme clcfi- (pcrformccl hy D r . Pctcr A . Warcl) ivcre all norm;~l.A hlono cicncics have hccn clcscrihccl. 'l'lie entity lias bccn :~ssociatccl spot test (Ortho) and fchrilc agglutirlins were negative. Stools ~
~
159
LEUKOCYTE ENZYhlATIC ABNOllhiALlTlES were negative for ova and p:lrasitcs. and blood irrc;i nitrogen, crentininc. blood sugar, alkaline phosphutnsc, scruni glutnmicoxaloacetic a n d serum glutamic-pyruvic transaniinases, a n d lactate clcliytlrogeniisc were all within normal limits. A t the time of referral. S ~ r r p l ~ ~ ~ l o c oc~pit1crttritli.s. c~(~r~.r Strc~l~rococcrts ~ ~ i r i d ~ t;iricI rs. g r o u p 1) P-hemolytic streptococcus were recovered from infected skin lesions. .l'ests of clclaycd skin hypersensitivity to m u m p s virus \\.ere positive hut negative t o tuhcrculin. cocciclioitiin. blastomycin. and 1iistopl:ismin.
NI:UI'ROPIIIL ENZYhlE
h1ATERIALS AND hlE'I't1OIlS SUBJECTS A n 1 I-year-old fernale and her family \\,ere stutlictl ;it Gcorgetown University IIospital (Table 1). Age-adjusted and adult subjects were inclutled a s control suhjects for the biochemical ant1 bactcricirlal studies clcscrihccl below. PRI~l'Allr\TION 01: LEUKOCYTE SUSPENSION hletliocls for the preparation of a relatively rich suspension of granirlocytes \\.ere those clcscribcd by Baehncr ; ~ n t lNathan (4) wit11 minor rnodific:ition. as previously clcscribcd ( 6 ) . Differential count of this lluitl revealeel values consistently i l l excess of 9 0 % polymorplionuclear leukocytes.
T h e clumititativc NL3.f tcst was performed by the method of Baehncr and Nathan (4). results being cxprcssccl as the cliango in optical density pli;~gocytosingvalues for 7 . 5 x 10" cells for 15 min at 37". Bi\C'I'I:RICIDAL
DE~I'ERhllNAl'IONS
Cell homogcnatcs were preparecl fro111 frozen cruclc cell suspensions by methocls descrihccl previously ( 6 ) . T h e sirpcrn;it;ints were analyzed for protein content (33) ancl the enzymatic assays were pcrformcd a s clescrihcd below. - 1 - 1 ~ ,ict~vitics .~ . ' of G-0-PD. 0-pIiospliogluc~)~iiitc tlchydrogcnasc (6-PGD), and NADP-specific glutathione reductase in white cell lysates \\,ere tlctcrmincd by the rnctliocl of Richterich ( 3 1 ) . Glutathione peroxitlase activity was determined by the method of Paglia a n d Valentine (26). T h e results of all leukocyte enzyme activities a r c reported a s nanornoles per min per rng protein a t 75". Leukocyte N A D P content was assayed according to the procetlure of Klingcnbcrg ( 2 2 ) and is rcportcd a s nnnomolcs per 15 min per 10' leukocytes. T h e optical densities were clctcrmined with a spectrophotomctcr using I-cm cuvcttes that were maintained at 25' by means of a circul;~tirigwater b a t h . G-&PI) LABILITY
A N D hllXlNG
EXPElllhlEN'I'S
Experiments were performed t o determine the effect of stabilizing agents o n G-6-PD activity during scri:~l incubation periods a t 37" accorcling t o rncthotls prcviously described (6). Leukocyte homogcnates, prep~lrcclfrom leukocytes frozen a t -70°, were tcstccl both in the absence ancl prcscncc of 6 pM N A D P plus 2 5 m h l 2-mcrcaptoethanol (2-hlE) a t 0, 3 0 , 6 0 , a n d 120 min of incubation at 37" (Tiible 3 , Fig. 2 ) . I n o r d e r to assess the possibility of a G-6-PD enzyme inhibitor, leukocyte homogenates from the patient and a normal control subject were rnixcd in equal proportions and lability studies were performed in accordance wit11 the methods just describccl. T h e results \\..ere compared with the lability stuclics of the patient's and the control's cells when tested individually (Fig. 3 ) .
TEST
CLINICAL DATA T h e hiictcriciclal assay was pcrformcd by the mcthocl of Quie (11. ( 7 9 ) using S~rr~~lr!~lococcr~.s (rrlr(,rl.s :IS a test strain. T h e Cli~iicaldata o n thc patient's family arc give11 in Table 1 a n d results were cxpressecl :IS the pcrccnt:ige of viable bacteria re- :~ccorclingt o the pctligree slio\vn in Figure 1 . It can be seen that, maining after 170 riiin of i ~ i c ~ r l x l t i cwith ~ ~ i p o I y r i i ~ ~ r p l i o ~ i ~ ~ c of I c :the ~ r 17 n i e ~ n h c r sfor \vliom information was available, infecleukocytes. tions were seen ill the maternal grc;itgranclrnothcr (11-2). the
c.1
Falllily mcrnhcr GG A1 GGF KA KC
No. in pc~ligrcc I- I I-? 11-1 11-7
Age. Ilclatioli to prohand -hlatcrlial grcatgr;inclinothcr hlatern;il grcatgr:illdfathcr hlaterlial grandfather hlatcrn;~lgranclmothcr hl;~tertial\tcpgr;illilf:ither Uncle by marriage hlutern;il aunt hlother
WI* C'C'
h1C
IV- l IV-2 1V-3
First cousin First cousin Brother
Scs F hl hl
Died 71 Ilicd 8 2 54
F
54
-
Age at onset of infcctions. years Unknown 0 0 44
Clinical symptornatology Skin infections. chronic bronchitis tfe;ilthy Healthy Cystitis. rash. rcpcatc~lb;~cterinl upper rcspiratory infections l lcnlthy Rlicumntic fever, hc;ilthy Chronic sinusitis, multiple allergies Eczcina, recurrent periodontal and urin;iry tract infcctions Allcrgics since chiltlh(~od Ilepcated pliary~~gotolisillitis, p;uicytopc~iia Ilcpcatcd bacterial pneumoni;~~ cliagriosed as liypogammaglob~~lincliiia ;ind treated with y-glohulin, age 7-6 years hlilk ;rllergy, sillusitis hlilk allergy, sinusitis. hordcolum h n~o-1 year Asthnl;~,recurrent pliaryr~goto~isillitis;ind otitis mcditi, urili:iry tract infcctions. boils 011 skin. granuloma removeel i i i 1967 from arm Ilcpeatccl pliaryngotol~sillitis;incl otitis rnc~li;i(see text) Atopic cczcnl;i, milk allergy, no recurrent infcc-
niothcr (111-3). ancl t \ \ o siblings (1V-3 ancl IV-4). A strong patielit ;~nclthe t u o ni;rlc silllings \vith impairetl NB'I' clye rccluchistory o f allergic clisc;~sc\ \ ; I S obtainccl in scvcr;ll family mcm- tion Iiacl significantly rcducecl G-0-I'll ;~ctivity\vlicn tcstccl i l l the I x r s (11-7, 111-7. 111-3. 111-4. IV- I , IV-2. IV-3. IV-0). A l t l i o ~ ~ g l i absence of N A I I P ant1 7-RIE at 37' (Tahlc 3 ) . .l'lic r e s ~ ~ l ot sf :I chilrlhoo~lhistory of liypogammagIoI>11li11c1iii:1 was oht:~inctlin Inore estcnsivc ilivcstig:~tion of G-0-1'1) Iahility o n the paticnt ancl the immccli:~te fc1mily itre slionn in Figi~rc3 . All extreme ;I matcrlial uncle (111-0). tests of this incliviclual's serum imniunolability o f Icukocytc G-0-PI> \vas observccl in the p ~ ~ t i c ncvcn t. globulin Icvcls tvcrc founcl to be \vitliin ~iorniallimits. v,Iicn tcstccl in the presence of 7-hIE anel N A I I P . G-6-1'1) lallility was not clctcctcd in any other farnilv members \\hen N13.r AN11 l3AC1'~:ls1
G-6-PD + NADP + 2-ME
GSSG rcduclase
6-PGD
GSH pcroxidase
Proband Father Mothcr Brother Brother Brother Maternal grandmother Maternal aunt 118.0 + 4.0 129.8 + 12.5 46.2 + 2.1 30.9 + 0.5 82.7 2 3.23 01= 37) (11 = 4) (n = 44) (11 = 26) (11 = 26) Enzyme activity values expressed as nanomoles per min per mg protein 5 SE mean. G-6-PD: glucose-6-phosphate dchydrogcnase; 2-ME: 2mcrcaptocthiinol; 6-PGD: 6-phosphogluconate dchydrogenase; GSSG: oxidized glutathionc; GSH: reduced glutathionc. No enzyme stabilizing agents added. 3tatistical significance compared to normal (Student's I-test); P < 0.005. P < 0.01. P < 0.025. Control
n
':v-
80
47
- -.--
K.C.
-:- -- _- - .
-1t -A*
\
Glycogen
B.L., M.
'S.E.
H202 (Bocleriol)
Glucose-Glucose-6-P
\control -- - - .------' N.L. ---4
Bactericidal Complex
W.L. 6-R~bulose j-Phosphate (Leukocylic)
a Glutathione peroxidase Loclale-Pyruvate
@
x'
K.L.
-
I
1
K r e b s Cycle
"0
90 120 Incubation Time (Minutes at 37OC) 30
60
Q N A D P H S p e c i f ~ cglutothione reductase Q Glucose-6-phosphate dehydrogenase @ N A D P H oxidase Q) 6-Phosphoglucanole dehydrogenase
@ N A D H oxidose
0 Phosphoglyceraldehyps
dehydrogenase
@ NADH-linked loclic dehydrogenase @ Catalose MPO=myeloperoaidose
Fig. 2. Leukocyte glucose-6-phosphate dchydrogcnase lability of patient ( K L) anrl her inimcdiatc family.
Control Mixing Experiment (Control t Patient)
K.L. (Patient)
s
OO
30 60 90 120 Incubation Time (Minutes at 3 7 O ~ ) Fig. 3. Leukocyte glucose-6-phosphate dchydrogcnase mixing cxperimcnt. glutathione peroxidase was first described in females with C G D (18), and subsequently in a male child with this syndrome (24). In contrast to the present studies, however, a lability of G-6-PD was not detected. Another two patients with C G D have been shown to have deficient leukocyte superoxide production (13). The iniportance of the superoxide radical is that it can be bactericidal itself, o r it may be further reduced to H,O,. The H,O, formed may react with additional ~noleculesof superoxide to
Fig. 4. Leukocyte cnzymc biochcniistry. form hydroxyl radicals o r nlay participate in the production of bactericidal aldehydes (32). A n iodination defect has also been described in the leukocytes of patients with C G D (21). I n the present studies the labile G-6-PD enzyme in the leukocytes of the two male siblings could be stabilized by the addition of N A D P and 2-ME, indicating the importance of this cofactor and of sulfhydral linkages in stabilization. The patient's G-6-PD was so severly labile, however, that stabilization was not possible even with the use of both of these agents (Fig. 1). The leukocyte G-6-PD lability in this family could be explained by the reduced content of leukocyte NADP, a deficiency of stabilizing factors, o r an altered N A D P to NADPI-I ratio as suggested by the studies of Erickson et a l . (15). This is consistent with the findings of Yoshida (35), which indicate that monomeric G-6-PD is linked together by a ~noleculcof NADP, forming an active dimer. Abnormalities in the glutathione system were also detected in this kindred (Fig. 1, Table 3). The finding of diminished activity of glutathione peroxidase is of interest because of the data of Strauss et a l . (33). These authors have suggested a primary role for the glutathione system in initiating the biochemical cascade resulting in the marked increase in HMP activity and H,O, production necessary for normal bactericidal activity. The deficiency in NADPH-specific glutathione reductase detected in the two male siblings with G-6-PD lability and in both parents further differentiates this particular syndrome of neutrophil dysfunction from the classic X-linked form in which an elevated activity of leukocyte glutathione reductase was reported (18).
In the franlc\vork of the schcm:~proposetl by KIcb:rnoff (90). 2. tlcpicting the inter-rclationsliips of the Icukocyte enzymes (17ig. 4). thrcc recent cornmunic;rtio~~s help to elucitlatc the pirthogcnctic nlcch:rnisrns involvctl in this kindred's tlisortlcr. 3. O n the lxrsis of results obtained using a grir~iulocytc-frcc 4. system in \\hich s:rnthinc oxitlasc is suhstitutccl for NAIIPfI oxitlase. Curnuttc (11. (1 1) speculated that the hulk of the 5. oxitlation that occurs \\hen NALII'FI. g r a n u l o q t c particles. ant1 m;rng:rnese ;ire illcuhatctl together takes plircc via :I noncnzy0. nlatic free r:rdical chain re;rction. 'fhcy concludecl that the function of NA1)I'Il oxitlasc \voultl he t o generate the supcrositlc rirtlical necessary t o initiate this reaction. Thcsc results arc pcrti7. ncnt in that these iruthors h;rve previously reported (1 7 ) a tlcfcct in the pyritlinc nuclcotitlc tlepcndcnt protluctioli of supcrosicle in the leukocytes of three p;rticnts n i t h X-linked C G I I . 'l'hey 8. favorctl NAIlI'I1 ositl;rsc as the tlcfcctivc cnzymc in thcsc patients. Ilohn antl Lehrer (16) have dcrnorlstrirtcd that the activ0. ity of NADPFI oxidasc in polynlorphonuclcnr Icukocytcs from patients with C G I I was 8% of normal. These authors conclutlecl 10. that the absence of a cyanide-insensitive N A D P H oxidasc o r a deficiency in its activntioli is the primary metabolic defect in the X-linked C G D syntlrome. O u r data is consistelit with both I I . of thcsc fillclings in that a tlcficiency in the N A D P H oxitlase system could result in reduced leukocyte N A D P content and the 12. G-6-PD lability. Since N A D P is a regulirtor of H h l P activity (5). its unavailability woultl inhihit the direct oxitlation of glucose through the HhlI' cithcr directly, o r secondarily by il1activ;rtion 13. of G-6-PD, accounting for the well known tlecreasctl H h l P activity in C G D leukocytes (3, 17). It is tempting to spcculirte that ;I dcfcct in the N A I I P generating system could :rlso be related 14. t o the tlccrcasetl activity of glutatliionc pcroxidasc, since this en- 15. zyme requires H,0, irs a substrrrte (Fig. 4). Thus, a clefcct in the NAIIPFI oxidasc system coulcl not only account for our biochemical findings, but also the inability of the leukocytes to I h . protlucc lI,O, supcroxitle, thereby causirlg a tlefect in their bac- 17. tericidal function. Although the :rffcctccl m;rlc mcmbcrs of this family have not hat1 fulrninant bilctcrial infectiolls. \vc feel that their clinical 18. manifestations arc compatible \vith the courses of patients prcviously tlcscribctl (8, 9 5 ) \vho h;rvc m:rllifestcd o rniltler clinicirl 1 0 . form of C G D . 'I'hc heterozygosity dcmonstr;rtctl in this kintlrcd is consistent with a n :rutosomal rcccssivc mode of tr;rnsrnission. 2 0 . 'l'hc patient. \vho manifests the most scvcrc clinical illness. 2 1 . u.oultl he l~omozygous;the unaffected male sibling. ~ \ l l I ant1 . the pirrcnts. NL ancl IVL. woultl he unaffcctctl carriers of the gcnc; ant1 the other two male siblings. 1)L and Ill,. \vo~lldbe hetcrozy- 22. gotcs with an intcrrnctliatc form of expression accounting for both their milt1 clinic;rl fi~icli~igs. their impairctl N B T tlyc rcduc- 2 3 . tion, ancl tlccrcascd Ixrctcricidal activity. T h e st~rtly of this kinclrcd has provirlcrl further cvitlcnce tliirt CGI) represents a 24. s11cctrt1111of Icukocytc enzyme itbnormalitics anti h:rs i~nplic;rtctl G-6-1'1) lability ;rntl the NAIII' gcrlcrating system :IS a n etiologic 25. firctor i l l the ncutropllil tlysfunction of this kintlrctl.
cytic C'cll in llost K c s ~ \ t a n c e lip. , 173-200 ( R ; ~ v c nPress. New Y o r k , 1075). Baehncr. R . L.. a n d Karnovsky, h l . L.: Deficiency of reduced nicotin;~mideadenine ilinuclcotidc oxidase in chronic gr;~nulornatouscliscase. Science, 162: 1 2 7 7 (1968). 13;lehncr. Ipsosim;ttcly 0 3 2 mOsm/liter. 13iliruhin-human alhurllirl solutions \ \ e r e prepared by clissolving I>iliruliin (Sigma) in 0 . 5 N N a O l l and adding the I>ilirullin t o a l l > u ~ i i i ~(crys~;tllinc, l C'11ttc.r) tli\\ol\rCd i l l 0 . 0 5 5 X I phosphate buffer, p1~17 . 4 , ionic strength 0 . 1 5 . All I>iliruliin solutions were l \ ~ r ~ acids i ~ ~ oill so111tio11(lo 11o1irl)1)c;ir t o i ~ ~ t e r f c vit11 r c ljiliru- p r c p ; ~ c c lanel maintai~icclin the cl;lrk o r sul~cluccllight. 1:in:tl p t 1 I I ~ Itr;~lrsport. I I';~re~iter:~l : r I i ~ ~ i c ~ ~ t : iwit11 t i o ~ ia r ~ ~ i r :rcicls lo S I I ~ I I I I I of bilirubin-all>l~mins o l u t i o ~ l swas 7 . 4 . I)c :I safe proccclurc ill j:~rrlrclicecl p:ltic~rts. 'l'llc intcractio~lof ;\mino a c i ~ l swith hilirul>in-all,unlin cornplcacs : r ~ l c l tr;~nsport\vcrc stueliccl by cotllpctitivc hincling :rssa!,s using c l i o l c s ~ r a n l i n e;trlcl Sepliaclcs gel filtration. the perosiclasc I'arcntcral fcccling is frcclucntly rcclr~il.c~l in prcm;tturc ancl assay. red cell upt:~hcof raclioactivc Ililirullin, ancl lly a n arialysis sick nc\\ horn infants \\.ho a r c ~ln;tl>lcto take a ~ l c c l t ~ a ~t el u t r i t i o n of :rbsorptio~l spcctr;r. I>y ~ n o u t h . I'hc p:~st tl~,c:~clch : ~ \ I11.ottglit tcc.litiic;~l ;icIv:~nccs \\ hich rnahc i t possible t o supply not only c;trl)ohy~lratc.I n i t ; ~ l \ o fat ;tnd a m i n o ;rciels by ;III in1r;tvcnou.; route. Uy these means. suslainctl gso\\th has h c c n ; ~ c l i i c v e ~inl 170th prcm:tturc ,inel term C'o~llpctitivc bincli~lg I ~ c t \ \ , c c ~a l l ~ u m i n a~lcl cholcstyramiric ( 1 2 ) \\as studiccl 11sir1ga ~ ~ r c i c c t l u rnloclil'ic~l e from Sch~lliclor ol. i1lf:111ts(2. 3 ) . 1Jnconji1g;ttctI I~ypcrl>iliruhincrni;~ i \ frcclucntly p s c s c ~ ~int sick ( 1 0 ) . I3ilirul>in-:tll~111iii11solutions \ \ e r e prcp;rrccl ,o cont;rin 5 gl I 0 0 1111 1111111;111sc1.11111 ;1lhurlli11;I ~ i l i r ~ r l ~ i ~ ~ / ; t l 111oI:rr l ~ ~ ~ ~ i ir;rtios r~ of 11c\vl>ornsrcccivi~lghigll c o n c c ~ l t r a l i o n sof a ~ n i n o;~citlsancl/or 0.5, 1 .O. 2 . 0 ; anel ;111lino;leiel c o r l c c ~ l t s ; ~ t i o f~ 0~ s. 0.SO. ;~ncl2 . 6 7 lipids. Silicc tllc possil7ility of kcrnictcrr~sis kn0\\11 t o he ing/lOOnll. /-\I) cclual volunlc of p r c \ \ ; ~ ~ l ~ c hc ol l e s ~ y r a ~ n i ~( 1l c2 ) crc;tsccl Iiy tllerapcutic agents \\ hich intcrl'crc \\it11 Ilinclirlg of c stirre11 c o ~ ~ \ t : l ~ ~ t i y . \ \ a s acl~lccl;111cl tllc ~ n i ~ t u s\v;rs bilirulii~lt o pla\nla ; ~ l l ) u ~ n (7. i n 1 I ). i t is inrportant t o c v a l u : ~ t c suspc.~~\irin /\licluol\ \ \ e r e rctno\.c.cl ;I{ 0 , 1 5 . 3 0 . 0 0 , ;11111 0 0 l l i i ~ lof C Y P O S ~ I ~ ~ o n hilisl~hir~ transport. tile cfl'cct of p ; ~ r c n t c r ; ~n li t t ~ . i c ~scilutio~is it ;11ic1I'illcr~cifree of 1.c5irr t l i s o ~ ~ g~h1 ; 1 \\\oo1. s ' 1 ' 1 1 ~e ~ ~ ~ e c ~ i l r ; t t i ~ ~ i 'l'llis stucly e ~ , a l ~ ~ atile t c s i~lflucnceof ;r c o ~ ~ l r n e r c i a l l;tv:ril;thlc y ;~l \v:I> c l c ~ c ~ - ~ ~ by ~i~iccl ~ L , I ~ I : Iit1 ~ Ii ~I i~c lIi I\ i~~ I ~ ti'iltr:~tcs lli~i t o 1111111:111 of l~iIir-t~l)i~i syntllclic :rmino acid soluliorl 0 1 1 I > i l i ~ - ~ ~hi~iclillg the ~nc,tllc>clof hlallo), ant1 Il\.cl!t~ (0). ('o~llrcilc s l > c r i ~ n c ~ iilll s scsuln ;1ll)1111li1i : I I ~ C I red h l o o ~ lcell\. 'l'llc effects of s y r ~ t l ~ e t arl~illo ic :~citls o n I)ilirrrl)i~itrarlsport \ \ e r e i l ~ ~ e s t i g a l c cnit11 l corlrl)ctiti\e I ) i ~ ~ c l iassays, ~ ~ g perosiclasc l i c recl cells, ii~rtl :Issay, isotol)ic sfuciics of I)ilirlrl)i~r~ ~ l ) t ; ~11.) clin'crence spectroscopy. licsults intlic:ltctl that a~rririo:lcitls llatl 110 sig~~ific:rl~t cl'fcct 011 tile c I i s t r i l ) ~ ~ t iof o ~l)ilir~rl)i~i i a t p i g ~ ~t ~ o c oll)urr~illrlrol:lr riltios liliclj t o I)c c~icourrtcretlill clirlical situatiorrs.
