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•a CLIN . CHEM . 21/1 . 93-9 7 (1975 ) 

# T^-mi

ZincProtoporphyri n (ZPP) : ASimple , Sensitive , Fluorometri cScreenin g Tes t fo rLea d Poisionin g
Angal o A . Lamola, 1 Morri s Joselow, 2 an d T . Yamane 1

Th e predominan t fluorescen t porphyri n tha t appear s i n erythrocyte s a s a resul t o f chroni c lea d absorptio n o r iro n deficienc yanemi a i s zin cprotoporphyrin

. Thi s com poun d ca n b e fluorometricall yassaye d i n dilute d whol e bloo d wiv h hig h sensitivity

. A n extremel ysimpl e an d rapi d tes t fo rlea d absorptio n i s describe d tha t i s base d o n thi s assay . Result s o f a fiel d tria l indicat e tha t thi s tes t shoul d b e seriousl yconsidere d a s a primary screenin g tes t fo rmonitorin g th e effect s o f chroni clea d absorption

. Additiona l Keyphrases : toxicology • trace

elements • iron-deficiency

anemia • porphyrias Th e massiv e campaign s no w underwa y i n thi s countr y t o detec t

lea dpoisonin g i n childre n hav e create da n urgen t

nee dfor apractica l screenin g test , on e tha t i s simple , reproducible , cheap , and , o f course , diagnosticall y valid
. Direc t analyse s o f

bloo d fo r lea di s seriousl y handicappe db y th e requirement s o f expensiv e equipment , skil l i n analytica l technique , and , abov e all , b y th e scrupulou s car e demande dt o avoi dth e ris k

o f sampl e contaminatio n fro mth e ubiquitou s lea di n th e environment
. I n recen t years , suc heffort s hav e instea dbee n di

recte dtowar dmeasuremen t of som e o f th e metabo

lite s (o r thei r associate denzymes ) resultin g fro mth e interferenc e b y lea dwit hhem e synthesis , suc ha s {-aminolevulini c acid , t$-aminolaevulinat e dehydra

tas e (E C 4.2.1.24) , o r

coproporphyri n (1, 2). Th e mos t likel y candidat e t o hav e emerge da s ausefu l "metaboli c sentine l o f

lea dpoisoning " i s extractabl e protoporphyri n i n bloo d(3-7).

1 Bel l Laboratories , Murra y Hill , N
. J
. 07974
. 2 Department s of

Preventiv e Medicin e an dPathology , Colleg e o f Medicin e an dDentistr y o f Ne w Jersey , Newark , N
. J
. 07103
. Receive dSept
. 18,1974 ; accepte dOct
. 15,1974
. Th e increas e i n "fre e erythrocyt e protoporphyrin " associate dwit hlea dintoxicatio n ha s bee n recognize d fo r mor e tha n 4 0 year s (8, 9). I t endow s erythrocyte s wit hare dfluorescenc e whe n excite dwit h blu e (40 0 nm ) ligh t (10), an di t

i s extractabl e fro mth e erythro

cyte s a s protoporphyri n wit hacidi c solvent s (11) (sucha s amixtur e of

ethy l acetate , aceti c acid , an d HCI)
. Th e extracte dprotoporphyri n ca n b e assaye d b y absorptio n spectrophotometr y or , wit hgreate r sensitivity , b y fluorometry
. Bot hth e presenc e of flu

orescenc e i n erythrocytes , observe dmicroscopicall y (12), an dth e presenc e of acid-extractabl e protopor
phyri n (3-7, 13-15) hav e bee n use da s monitor s o f th e effect s o f

lea dintoxication , bu t

neithe r tes t i s highl y specific : a n increas e i n th e protoporphyri n and , concomitantly , i n th e re dfluorescence , i s als o associate dwit hsevera l othe r disorders—erythropoi

eti c protoporphyria , iro n deficienc y anemia , an dhe

molyti c anemia s (13, 16, 17). Furthermore , th e rela

tionshi p betwee n th e concentration s o f th e protopor
phyri n an do f

lea di n bloo dan dth e factor s governin g tha t relationshi p ar e a s ye t

unclear
. Apparentl y overlooke di n th e flurr y o f reporte d studie s (3, 6) o n th e us e of erythrocyt e protoporphy

rin s ar e som e o f

th e basi c chemica l an dspectrofluo

rometri c aspect s of

th e porphyrins
. Recen t result s concernin g th e natur e o f th e fluorescenc e exhibite d b y th e iron-fre e protoporphyrin s o f erythrocytes 3, 4 —easil y detectabl e i n th e hemoglobi n fractio n o r th e whol e blood—lead s u s t o propos e th e us e o f amuc h simple r procedur e base do n zin c protoporphyrin 3 (18) fo r detectin g person s affecte db y lea dintoxica

3 Lamola , A
. A. , Yamane , T. , Piomelli , S. , Poh-Fitzpatrick
. M. , an dHarber , L. , unpublishe dresults
. 4 Piomelli , S. , Lamola , A
. A. , Poh-Fitzpatrick , M. , Carlos , E. , Seaman , C , an dHarber , L. , unpublishe dresults
. D : Oi 

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. 1,197 5 9 3P. 3

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1 1 1 1 i l l ~"i — 56 0 58 0 60 0 62 0 64 0 66 0 68 0 70 0 72 0

WAVELENGT H (nm ) Fig

. 1

. Fluorescence spectr a o f 400-fol d dilute d whol e bloo d Th e dashed

curve i s th e spectru m obtaine d fro m bloo d o t patient s wit h lea d intoxicatio n (Pbl). Th e solid curve i s th e spectru m obtaine d fro m patient s wit h erythropoieti c protoporphyri a (EPP). Th e tw o mai n band s i n eac h spectru m ar e th e 0 ' - * 0 an d 0 ' - » 1 vibrationa l transition s associate d wit h a singl e porphyri n emissio n an d d o no t indicat e that tw o type s of porphyri n ar e fluc -

. rescin g i n eac h case . Th e emissio n fro m Pb l bloo d i s tha t of zin c protoporphyrin . Th e mai n fluorescenc e fro m EP P bloo d i s characteristi c of metal-fre e protoporphyrin . EP P bloo d usuall y shows som e zin c protoporphyri n fluores cenc e (th e shoulde r nea r 59 0 nm )
tion
. Thi s compound , th e fluorescent for mof proto
porphyri n associate dwit hlea dpoisonin g (18), ca n b e measure ddirectl y an dsimpl y i n dilute dwhol e blood , an dit s concentratio n ca n b e directl y relate dt o hig h lea dconcentration s i n th e bloo dof bot hchildre n an d adults
. Method s an d Material s Analytica l Principle s Whe n bloo dsample s fro mpatient s wit herythro

poieti c protoporphyria , lea dintoxication , o r iro n de

ficienc y anemia , ar e aci dextracte di n th e usua l man ne r (6) th e fina l hydrochlori c aci dsolution s exhibi t identica l fluorescenc e spectr awit hamaximu ma t 60 6 nm 5 (th e excitatio n maximu mi s nea r 40 5 nm)
. Thi s well-know n spectru mi s characteristi c o f protopor
phyri n I X i n acidi c aqueou s solutions
. I n contrast , th e fluorescenc e spectru mof whol e bloo do r

th e he

moglobi n fractio n fro mpatient s wit herythropoieti c protoporphyri ai s quit e differen t fro mtha t

obtaine d fro mth e bloo do r

hemoglobi n o f

th e latte r

tw o cate
gorie s o f patients 3 (18). Th e spectr aar e show n i n Figur e 1
. Thei r

bloo d[dilute d400-fol dwit hsalin e ( 9 gl/liter)-phosphat e buffe r (1 0 mmol/liter , p H 7.4) ] o r hemoglobi n exhibit s afluorescenc e maximu ma t 62 5 n m(excitatio n maximum , 39 7 nm)
. Thi s fluorescenc e i s attributabl e t o protoporphyri n I X i n it s free-bas e form , boun dt o hemoglobi n a t a n unknow n site(s). 3 I n contrast , th e whol e bloo do r hemoglobi n fro m lead-intoxicate do r iron-deficienc y anemi c patient s fluorescenc e maximall y a t 59 4 n m(excitatio n maxi

mum , 42 4 nm)
. Thi s fluorescenc e i s du e t o zin c proto
porphyri n I X (ZPP) , whic hprobabl y occupie s site s of " hem e i n asmal l fractio n of th e hemoglobin ; i.e. , th e fluorescen t

porphyri n i n bloo dfro mthes e tw o kind s o f patient s i s no t

"fre e porphyrin" 3 (18 ). Blood s con tainin g uroporphyrin s o r coproporphyrins , o r

both , suc ha s thos e obtaine dfro mpatient s with porphyri a cutane atard aexhibi t

fluorescenc e maxim aa t wave length s greate r

tha n 61 5 nm. 6 Thus , b y avoidin g aci d extractio n of

th e blood , th e spectrofluorometri c mea suremen t o f ZP P allow s on e t o differentiat e easil y be

twee n lea dintoxicatio n an diron-deficienc y anemi a an dth e porphyrias , eve n erythropoieti c protopor
phyria. 3, 6 Curren t

procedure s fo r

measuremen t

o f th e fre e erythrocyt e protoporphyri n d o no t permi t thi s differentiation
. Spectrofluorometr y o f

ethano l o r

aceton e extract s o f whol e bloo dals o allow s differentiatio n betwee n lea dintoxicatio n an dth e porphyrias 3 (18). Ethano l o r aceton e extract s th e protoporphyri n fro mth e bloo do f leadrintoxicate dperson s togethe r wit h it s chelate dzinc
. ZP P i n ethano l o r aceton e fluoresce s a t 589 , wherea s protoporphyri n i n ethano l o r aceton e (e.g. , extracte dfro mprotoporphyri c blood ) fluoresce s a t 63 2 nm . However , th e efficienc y wit hwhic h etha

no l extract s thes e porphyrin s fro mwhol e bloo d varies—i t

i s 50-80%
. Extractio n wit haceton e i s eve n les s efficient
. T o increas e th e efficienc y o f extractio n wit hacetone , Chisol me t al
. (3,19) use dacidifie dac etone
. Whe n thi s i s use dt o extrac t lead-containin g blood , amixtur e of

ZP P an dfre e protoporphyri n i s obtained , becaus e thi s aci dsolven t ca n remov e th e zin c fro mZPP
. Thu s th e us e o f organi c solvent s i n suc hanalyse s for

bloo dporphyri n i s evidentl y mor e complicate dan dles s quantitativ e tha n simpl y per formin g spectrofluorometri c measurement s o n dilut

e dwhol e blood
. Apparatu s Fluorescenc e wa s measure dwit haHitach i Perkin

Elme r MPF 2 spectrofluoromete r equippe dwit h a Hamamats u 44 6 U Rtube
. Wit hthi s instrument , th e fluorescenc e o f

unextracte dporphyrin s i n whol e bloo dcoul db e measure ddow n t o norma l concentra
tion s (50-8 0 fig/liter). Wavelengt hmaxim aca n b e measure dt o ± 1 nm . Othe r commerciall y availabl e in

strument s suc ha s th e Aminco-Bowma n an dFarran d MK- 1 spectrofluorometers , equippe dwit h asimila r photomultipHe r

detector , ar e sufficientl y sensitiv e an dresolv e wel l enoug ht o giv e comparabl e data
. Lea di n whol e bloo dwa s determine dwit haPer

kin-Elme r

Mode l 40 3 atomi c absorptio n spectropho

tomete r equippe dwit hadeuteriu mbackgroun dcor rector
. Th e 283.3-n mresonanc e wavelengt hwa s use d throughout
. 5 Thi s refers t o th e maximu m of th e lowes twavelengt

h ban d i n th e spectrum , as measured wit h ared-sensitiv e photomultipHer tub e a s th e detector . 6 Lamola , A . A. , and Poh-Fitzpatrick , M. , unpublishe d results .

tfHfHWf tf^MfaP. 4

Procedur e Becaus e th e spectrofluoromete r tha t w e use dde

tect s fluorescen t

ligh t a t a n angl e of

90 ° t o th e direc

tio n o f th e excitin g light , th e bloo dwa s dilute d500

- fol dt o avoi dth e stron g absorptio n fro mnorma l he

moglobin
. Thi s dilutio n lyse dth e cell s an ds o als o serve dt o minimiz e ligh t scattering
. Scatterin g wa s furthe r reduce db y addin g 2 0 g of anonioni c deter

gent , dimethyldodecylamin e oxid e ("Ammony x LO" ; Ony x Chemica l Co. , Jerse y City , N
. J. ) pe r liter , whic himmediatel y dissolve s th e cel l stroma
. Thi s dilutio n wa s effecte db y addin g 2 0 pA of blood , obtaine deithe r

directl y fro mafinge r

punctur e int o a heparinize dmicrohematocri t tub e (Clay-Adams ) o r b y mean s o f

a n Eppendor f

pipe t fro mbloo dcollecte d b y venipunctur e int o heparinize dVacutainer s (Bec

ton-Dickinson) , t o 1 0 m l o f phosphat e buffe r

(1 0 mmol/liter , p H 7.4 ) containin g th e detergent
. Whe n s o diluted , th e fluorescenc e of th e resultin g solutio n remain s constan t

for a t leas t aweek
. Eve n if

whol e bloo di s store dfo r

aweek i n th e refrigerato r

befor e bein g dilute dwit hth e detergen t solution , th e mea sure dfluorescenc e of

th e ZP P i s no t

significantl y af

- -fected
. Th e fluorescenc e spectru mof eac hdilute dbloo d sampl e wa s recorde dwit hth e excitatio n monochro

mete r se t a t 42 4 nm . Th e intensit y o f th e spectru mof 59 4 n m(scatterin g backgroun dsubtracted ) wa s mea sure di n arbitrar y units , whic hwer e converte dt o concentratio n o f

ZP P (i n j*g/10 0 m l o f

blood ) wit h th e comple x o f

zin c protoporphyrin 7 an dapohemo

globi n use da s astandard. 8 Th e latte r material , pre pare da s describe db y u s (18), wa s assaye dspectro
photometricall y wit hus e o f th e valu e 1.2

2 X 10 5 fo r th e absorptivit y a t

42 3 n m(20). Fo r

day-to-da y ref

erencin g o f

fluorescenc e intensit y data , th e spectro
fluoromete r

wa s calibrate dwit hacoproporphyri n I standar d(Sigm aChemica l Co.)
. Th e procedur e o f

Hesse l (21 ), a s modifie db y Jose

lo w an dBogde n (22), wa s use dt o determin e lea di n whol e blood
.

* Zin c protoporphyri n I X ma y b e purchase dfromPorphyri n Product s (Dr
. Bruc e Burnham) , P.O
. Bo x 31 , Logan , Uta h 84321
. s I n orde r t o facilitat e wor

k i n othe r laboratorie s w e hav e devel

ope da s asecondar y standar dzin c protoporphyri n 0.0 5 ng/ra\ i n pyridine
. Th e latte r

solutio n ma y b e prepare da s follows : Dissolv e zin c protoporphyri n i n pyridine ; adjus t th e absorbanc e a t

42 5 n m t o 1.0/cm ; dilut e 100-fol dwith pyridine
. Th e fluorescenc e spec tru mo f thi s solutio n i s very similar t o tha t of th e lead-intoxicatio n bloo di n dilut e detergen t

solution
. Th e concentratio n o f ZP P i n th e dilute dbloo dma y b e determined , b y usin g thi s standar dan dth e formula : Z P P (Mg/10 0 m l blood ) = 1.3 4 x 5 x d I
. wher e I i s th e fluorescenc e intensit y (59 4 nm ) o f th e dilute dbloo d sample , I , i s th e fluorescenc e intensit y (a t 59 4 nm ) o

f th e standar d obtaine dunde r

identica l condition s (excitatio n 42 5 nm) , 5 i s th e concentratio n (/ig/10 0 ml ) o f ZP P i n th e standar dprepare da s di

recte dabove , di s th e bloo ddilutio n facto r (50 0 fo r 2 0 u\ bloo ddi

lute dint o 1 0 ml buffere ddetergent) , an d1.34 i s th e calibratio n facto r whic hwe hav e determine dbase do n th e zin c protoporphy

rin-apohemoglobi n standard
. 

# i

1 0 Z

O 30 4 0 5 0 6 0 7 0 6 0 9 0

Pb (ug/IOOm l b>ood ) Fig

. 2

. Relatio n betwee n fluorescenc e intensit ya t 59 4 n m o f dilute d whol e bloo d an d th e whol e bloo d lea d concentratio n Dat a ar e fo r bloo d fro m 25 0 youn g childre n (small filled circles) an d 3 5 me n (larger open circles). Th e fluorescenc e intensit y unit s wer e chose n t o reflec t (withi n 10% ) th e concentratio n of zin c protoporphyri n (ZPP ) a s /i g ZP P per / 10 0 m l of blood . Th e uppe r lin e i s th e regressio n lin e fo r a linea r fi t ( r = 0.77 ) o f th e childrens ' data : i t represent s th e equatio n lo g ZP P Oig/10 0 ml ) = 0.5 2 + 0.02 7 P b (/jg/10 0 ml) . Th e lowe r lin e i s th e regressio n lin e (r = 0.87 ) fo r th e dat a fo r th e adults : lo g ZP P (jig/10 0 ml ) = 0.5 4 + 0.01 7 P b (j.g/10 0 ml) . (N o mode l ha s bee n presente d t o justif y a linea r relationshi p betwee n th e lo g of th e porphyri n fluorescenc e an d lea d concentratio n an d w e d o no t conside r thi s semilo g plo

t t o b e th e most reasonabl e wa y t o displa y th e relationship . However , other s (6, 14) hav e fitte d simila r dat a i n thi s wa y an d w e d o s o s o that ou r result s ma

y b e bette r compare d wit h theirs. )

Th e fluorometri c measurement s wer e performe da t Bel l Laboratories , th e lea ddetermination s a t th e Colleg e of

Medicin e an dDentistr y of Ne w Jersey
. Th e stud y wa s don e i n th e usua l double-blin dman ner . Th e specimen s wer e receive dfro mchildre n wh o wer e takin g par t

i n ascreenin g progra mi n th e New

ark , N
. J
. area . Th e specimen s fro mme n wer e ob

taine dfro marando mgrou p of worker s a t

asmeltin g plant
. On e fluorometri c tes t an don e lea ddetermina

tio n wa s mad e o n eac hspecimen
. Al l th e dat awer e use di n th e statistica l analysis
. Result s A semilo g plo t

o f fluorescenc e intensitie s vs . lea d concentration s fo r

25 0 bloo dspecimen s fro mchildre n (ages , 1 % t o 5 years ) i s show n i n Figur e 2 , alon g wit h simila r observation s fo r

3 5 me n occupationall y ex pose dt o lea di n asmeltin g plant
. Th e plo t suggest s tha t

th e lo g o f

porphyri n fluores

cenc e a t 59 4 n man dlea dconcentratio n ar e linearl y related
. Thi s i s simila r

t o th e patter n observe db y other s wh o hav e measured , a t differen t wavelength s (3-7, 13-15), th e porphyrin s extracte db y variou s technique s fromblood
. Moreover , th e fluorescenc e associate dwit h th e bloo dspecimen s fro mth e me n i s considerabl y les s

CLINICA L CHEMISTRY , Vol . 21 , No . 1 . 197 5 9 5P. 5

tha n tha t

foun di n th e bloo dof childre n wh o ha d comparabl e lea dconcentrations
. I n th e concentratio n rang e o f 40-6 0 Mg/10

0 ml , adul t

bloo dma y sho w fro m % t o % th e porphyri n fluorescenc e exhibite db y chil

dren' s blood
. Discussio n Regardles s o f

th e validit y o r

logi c of th e approach , aquantitativ e tes t

intende dfo r

us e i n mas s screenin g wil l requir e th e designatio n o f som e numbe r

a s acut
of f point , alimitin g valu e belo w whic hfurthe r actio n i s no t indicated
. Thes e number s hav e generall y bee n se t empirical

ly , usuall y afte r

considerabl e statistica l manipula

tion s tha t

d o littl e t o enhanc e th e strengt hof th e di

agnosti c test , an dwhic heventuall y involv e ajudg

men t o f benefi t vs . risk
. Th e benefi t

t o b e sough t

i s th e wides t

screenin g coverag e a t

th e lowes t

possibl e cost ; th e ris k

t o b e avoide di s th e occurrenc e of fals e negatives
. Tabl e 1 show s th e numbe r o f fals e negative s (i.e. , childre n wh o hav e som e degre e o f lea dpoisoning , bu t woul db e missed ) fo r

severa l bloo dlea dconcentra
tion s an dfluorescenc e cut-of f value s encountere di n thi s "study o f

unselecte dspecimens
. W e selecte daconservativ e rang e o f fluorescenc e cutof f values , becaus e o f

th e extrem e simplicit y an d lo w cos t o f th e test , a n approac hals o warrante db y th e limitation s of

th e test
. Despit e th e sensitivit y availabl e throug hmeasuremen t of fluorescence , sev era l indirec t

factor s commo n t o al l test s tha t depen d o n aporphyri n measuremen t predispos e towar dinac

curac y an duncertainty : 1
. ZPP , eve

n mor e tha n lead , i s associate dpredominantl y wit herythrocytes
. Therefore , strictl y speaking , th e whol e bloodfluorescence intensit y shoul db e correcte dfo

r the v hematocri t

o r hemoglobi

n valu e (3, 6), bu t

suchacorrec tio n woul db e significan

t onl y whe n mor e than moderate anemi ai s present
. Correctio n o f our value s fo r th e hemato

cri t made n o significan

t differenc e wit

hrespec t t o ascreen in g application
. 2
. Th e appearanc e o

f protoporphyri n fluorescence i s a delaye dphenomenon relate dt o erythropoiesi s an doccurs som e tim e afte r th e individua

l ha s been exposedt o chroni c absorptio n o

f lea d(4 ). Thus , achild' s bloo dma y possibl y sho w ahigh lea

dconcentratio n (a s aresul t o f aver y recent ingestio n o

f aleade dpain t

chip , fo r example ) an dstil l b e lo w in porphyri n fluorescence
. 3
. A n iron-deficiency anemia , a s note dearlier , ma y als o resul t in bloodwit

hazinc porphyri n fluorescenc e (18 ). I n a n inner-cit y populatio n o f children , prevalenc e o f iro n deficienc y anemi ama y b e a s hig ha s 10% 9 an d wil l hav e som e effec t

upo n th e bloo dlead-ZP P corre lations
. O n thi s accoun t

alone , however , th e ZP P tes t wil l als o offer

th e additiona l virtu e o f detectin g chil

dre n i n nee dof

medica l attentio n fo r

reason s othe r tha n lea dintoxication
. I t woul dalmos t

b e mandator y i n ascreenin g progra mt o loo k for a n iron-deficienc y anemi aamon g thos e childre n wh o sho w a n initia l Tabl e 1

. Fals e Negative s Observe d a t Severa l Fluorescenc e Values " Lea d concentration , i.g/10 0 m l 24 0 &5 0 26 0 Bloo d ZPP.M9 /

- 100 m l Proportio n % Proportio n % Proportio n % 1 5 3/25 0 1. 2 0/25 0 0 0/25 0 0 2 0 9/25 0 3. 6 0/25 0 0 0/25 0 0 2 5 14/25 0 5. 6 2/25 0 0. 8 0/25 0 0

•Bloo d lea d concentration s i n childre n hav e becom e arbi traril y divide d int o severa l categorie s i n accordanc e wit h recom mendation s o f th e Publi c Healt h Servic e (2i) a s wel l a s genera l practice : (a ) thos e les s tha n 4 0 pg/10 0 m l bloo d ar e a t presen t considere d acceptabl e o r no t actionable , eve n thoug h undesir able ; (6 ) thos e betwee n 40 an d 50pg/10 0 m l indicat e excessiv e absorption , a caus e fo r concern , an d generall y thos e abov e 5 0 Mg/10 0 m l invoke a recommendatio n fo r immediat e follow-up ; (r ) concentration s greate r tha n 60/.g/10 0 ml , i n man y healt h juris dictions , ar e considere d a s diagnosti c fo r lea d poisoning , an d hospitalizatio n ma y follow . An y bloo d lea d poncentratio n >4 0 fig/10 0 m l ca n thu s b e considere d a s a positiv e finding .

9 Piomelli , S. , an dChisolm , J
. J. , Jr. , persona l communications
. hig hZP P valu e an daconfirme dlo w bloo dlea dcon centration
. Despit e th e foregoin g qualifications , th e simplicit y of th e ZP P tes t

warrant s it s seriou s consideratio n a s a primar y screenin g test
. I f acut-of f fluorescenc e valu e of 1 5 i s set , the n fro mthi s stud y w e woul dpredic t tha t al l lea dvalue s greate r

tha n 5 0 jtg/10 0 m l wil l b e discovered ; an dtha t

les s tha n 2 % of

thos e childre n wit habloo dlea dconcentratio n of 4 0 Mg/10

0 m l o r mor e woul db e missed
. O n th e othe r hand , abou t

25 % of th e screene dchildre n ma y sho w fals e positives
. Thes e childre n woul db e reteste ddirectl y fo r bloo d lead
. Becaus e fluorescenc e ca n b e measure dquickl y (withi n afew minute s afte r specime n collecting) , suc haretes t

nee dno t involv e recallin g th e chil dan d th e complication s o f

schedulin g an dfollowin g throug ho n are-appointment
. I f ahighe r

cut-of f valu e (2 0 instea do f

15 ) wer e used , asmalle r

proportio n of

fals e positiv e (abou t 17% ) woul db e observed , bu t asomewha t

large r pro portio n (abou t

4% ) o f childre n wh o migh t

b e i n nee d o f medica l attentio n woul db e missed
. Thi s migh t b e considere dto o hig haris k t o tak e for

th e benefi t

(i.e. , th e fewe r fluorescenc e tests ) realized
. Beside s bein g asimple r

test , th e ZP P fluorescenc e assa y o n whol e bloo dshoul db e inherentl y mor e ac curat e tha n ar e thos e test s tha t involv e a n extractio n step
. N o extractio n procedur e i s use dhere , s o th e di

rec t measuremen t

canno t b e artifactuall y lo w be

caus e of incomplet e extraction
. Th e ZP P tes t

ca n b e simplifie deve n furthe r b y us e o f afluoromete r

tha t i s buil t wit h"fron t

surface " op tics
. Fo r th e procedure s describe dabove , dilutio n o f th e bloo dwa s necessar y onl y becaus e of th e righ t angl e optic s o f

th e spectrofluorometer s mos t com monl y availablerWit h "fron t surface " optics , fluores

cenc e ca n b e measure ddirectl y o n adro p of bloo do n 9 6 CLINICA L CHEMISTRY , Vol . 21 . No . 1 . 197 5P. 6

aglas s slide
. A small , inexpensiv e portabl e instru

men t wit hsuc hadesig n ha s bee n constructe db y J
. Eisinge r an dW
. E
. Blumber g a t Bel l Laboratories , an di s currentl y undergoin g fiel dtesting
. Thi s instru

men t shoul dmak e i t

possibl e t o examin e childre n i n high-ris k population s ofte n enoug ht o substantiall y increas e th e pubi c healt hbenefit s derive dfro mthi s test
. I n summary : ZP P ha s bee n demonstrate dt o b e on e o f th e metabolite s tha t

accumulat e i n whol e bloo da s aresul t o f lea dinterferenc e wit hhem e synthesis
. Th e direc t measuremen t

i n whol e bloo do f

th e fluores

cenc e attributabl e t o ZP P offer s asimple , sensitive , specifi c an dvali dtes t

fo r lea dintoxicatio n o r

a n iron-deficienc y anemia
. Othe r porphyrin-relate ddis

orders , suc ha s erythropoieti c protoporphyri ao r por phyri acutane atard aca n b e detecte di n asimila r way . Th e validit y an dusefulnes s o f porphyri n measure ment s a s diagnosti c criteri aof undu e lea dabsorptio n hav e bee n demonstrate db y th e studie s of Piomell i (5-7), Chisol m(3), an dGranic k an dSass a(13, 14) an dthei r

coworkers
. Th e extrem e simplicit y an d rapidit y of

ou r tes t warrant s it s consideratio n a s a primar y screenin g tes t

for monitorin g th e effect s o f lea dabsorption
.
W e than kS . Piomelli , J . Eisinger , and W . E . Blumberg forhelp fu l discussions , and Velm aHinch forexcellen ttechnical assis tance . W . E . Blumberg aide d importantl y in datahandling . Reference s 1 . Baloh , R. W. , Laborator y diagnosi s of

increase d lead absorp tion . Arch. Environ. Health 28,19 8 (1974) . 2 . Chisolm , J . J. , Screenin g technique s forundu e lead exposure i n children : Biological and practical considerations . J. Pediat. 79,719* * (1D71) . 3 . Chisolm , J . J. , Mellits , E . D. , Keil , J . E. , and Barrett , M . B. , A simpl e protoporphyri n assay microhematocri tprocedure a s a screenin g techniqu e forincrease d lead absorptio n i n youn g chil dren . J. Pediat. 84,49 0 (1974) . 4 . Chisolm , J . J. , Screenin g forlead poisonin g i n children . Pediatrics 51,28 0 (1973) . 5 . Piomelli , S. , (a ) Micrometho d fo rfre e erythrocyte porphyrins . Clin. Res. 70 , 497 (1972) ; (6 ) A micrometho d fo rfre e erythrocyte porphyrins : Th e FE Ptest . J. Lab. Clin. Med. 81,93 2 (107:.) . 6 . Piomelli , S. , Davidow , B. , Guinee , V. , e tal. , Th e FE P(free erythrocyte porphyrins ) test : A screenin g micrometho d fo rlead poisoning . Pediatrics 51,25 4 (1973) . 7 . Piomelli , S. , Davidow , B. , an d Guinee , V . F. , Lea

d poisoning : Statu s of th e FE Pand othe rtests . Pediatrics 52 , 46 8 (1973) . Let te rt o th e Editor . 8 . Va n De n Bergh , A . A . H. , and Grotepass , W. , Porphyrinami e ohn e Porphyrinuri aKlin. Wochenschr. 12 , 58 6 (1933) . 9 . Watson , R

. J. , Decker , E. , an

d Lichtman , li . C , Hematologi c studie s of children wit

h lead poisoning . Pediatrics 21,4 0 (1958) . 10 . Vanotti , A. , and Delachaux , A. , Iron Metabolism and Its Clinical Significance. Grune , New York , N . Y. , 1949 . 11 . Schwartz , S. , Berg , M . H. , Bossenmaier , I. , an d Dinsmore , H. , Determination s o f

porphyrins i n biologica

l materials . I

n Methods of Biochemical

Analysis, 8 , D . Glick , Ed. , Interscience , Ne w York, N.Y. , 1960 , p p 253-255 . 12 . Whitaker , J . A. , and Vietti , T . J. , Fluorescenc e o f th e erythro cyte s i n lead poisonin g i n children : An aid t o rapid diagnosis . Pediatrics 24,73 4 (1959) . 13 . Granick, S. , Sassa , S. , Granick, J . L. , e tal. , Assay s forporphy rins . Proc. Nat. Acad. Sci. U. S. 69,238 1 (1972) . 14 . Sassa , S. , Granick, J . L. , Granick, S. , e tal. , Studie s i n lead poi soning . Biochem. Med. 8,13 5 (1973) . 15 . Kammholz , L . P. , Thatcher , L . G. , Blodgett , F . M. , and Good , T . A. , Rapi d protoporphyrin quantitatio n fordetectio n o f lead poi soning . Pediatrics 50,62 5 (1972) . 16 . Marver, H . S. , and Schmid , R. , In The Metabolic Basis ofInherited Diseases, 3r

d ed. , J . B . Stanbury , J . B . Wyngaarden , and D . S . Fredrickson , Eds . McGraw-Hill , Inc. , Ne w York , N . Y. , 1972 , p p 1087-1140 . 17 . Heilmeyer , L. , Disturbances in Heme Synthesis. C . C Thom as , Springfield , 111.

, 1966 . 18 . Lamola , A . A. , and Yamane , T. , Zinc protoporphyrin in th e er ythrocyte s o

f patient s wit

h lead intoxicatio n and iron deficienc y anemia . Science 186,93 6 (1974) . 19 . Chisolm , J . J. , Jr. , Hastings , C . W. , and Cheung , D . K . K. , Mi crophotofluorimetri c assay forprotoporphyrin i n acidifie d aceton e extracts of

whol e blood . Biochem. Med. 9,11 3 (1974) . 20 . Leonard , J . J. , Yonetani , T. , and Callis , J . B. , A fluorescenc e stud y of hybrid hemoglobin s containin g free bas e zin c protopor phyrin . Biochemistry 13,146 0 (1974) . 21 . Hessel , D . W. , A simpl e and rapid quantitativ e determinatio n oflea d i n blood . At. Absorption Newslett. 1, 55 (1968) . 22 . Joselow , M . M. , and Bogden , J . D. , A simplifie d micrometho d forcollectio n and determinatio n o f lead i n blood usin g apape r disk-in-Delve s Cup technique . At. Absorption Newslett. 11 , 9 9 (1972) . 23 . Medica

l aspects o

f childhoo d lead poisoning . Th e Surgeon General' s policy statement , U . S . Publi c Healt h Service . Pediatrics 48,464(1971) . i t * 

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