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Murad . F. . Chi . Y.M. . Rail , T.W. . an d Sutherland . E.W . (1962) . J. Biol Chem. 237:\2J). Mylroie , A.A. , Moore . L. . Olyai , B. . an d Anderson . M . (1978) . Environ. Res. 13:51. Nathanson . J.A. . an d Bloom . F.E . (1975) . Nature 233:419. Nathanson . J.A. . an d Bloom . F.E . (1976) . Molee. Pharmacol. I2:i90. Nomiyama , K. . Sato , C . an d Yamamoto , A . (1973) . Toxicol. Appl. Pharmacol. 24:625. Pastan . I.H. . Johnson . G.S. , an d Anderson , W.B . (1975) . Ann. Rev. Biochem. 44:491. Peaslee , M.H. , an d Einhellig , F.A . (1977) . Experlentia 33 :\206 Peg? . A.E. , an d Williams-Ashman . H.G . (1968) . Biochem. J. '09.32P . Pentschew

, A. , an d Garro . F . (1966) . Acta Neuropathol. (J:266 . Perkins . J.P . (1973) . P. Greengar d an d G:A . Robiso n (Eds) . I n "Advance s i n Cycli c Nucleotid e Research. " Vol . J , p . I . Rave n Press , Ne wYork . Piscator , M . (1964) . Nord. Hyg. Tidskr. 43:16. Pohl , SL , Birnbaumer . I.. . an d Rodhcll , M . (1969) . Science 164:566. Raina . A. , an d Jannc . J . (1975) . Slid. Biol. 5J.I2I . Russell . D.H. , and Potyraj . J.J . (1972) . Biochem. J. 128] 109 . Russell , D.H. . an d Snyder . S.H . (1968) . Prtic. Nat. Acad. Sci. IU.S.A.) 60:1420. Sassa . S. , Granick , S. , an d Kappas . A . (1975) . Ann. N.r. Acad. Sci. !44:4\4. SaucrhofT. M.W. . an d Michnelson . I . A . (1973).. Scienc e I.12.W22. SchardiiiB , N.N. , an d Oehme , F.W . (1973) . Clin. Toxicol. <:4I9 . Singhal , R.L. . Kaccw, S . Sutherland , D.J.B , an d Telli . A H (1973) . ResCommun. Chem. Pathol. Pharmacol. 6:951. Singhal . R.L , Mcrali . Z. , Kacew, S. . an d Sutherland . D..I.B . (1974) . Science J.13:\094. Sirovcr , M.A. , an d l.ocb . L.A . (1976) . Biochem. Biophys. Res. Commun. 70.812 . Stevenson . A. . Mcrali . Z.. Kaccw. S. , an d Singhal . R.L . (1976) . Toxicology 6:265. Stevenson . A.J. , Kacew . S. , an d Singhal , R.L . (1977a) . Toxicol. Appl. Pharmacol. 40.161 . Stevenson . A.J. . Kacew. S. , an d Singhal , R.L . (1977b)./ . Toxicol. Environ. Health 7.1125 : " " Stoll . RE. . White . J.F. , Miya . T.S. . an d Bousquct. WF . (1976) . Toxicol. Appl. Pharmacol. 37:61. Sutherland . E.W. , an d Robison . G.A . (1969) . Diabetes 18:191. Vallee . B.L. . an d Ulmer . D.D . (1972) . Ann. Rev. Biochem. 47:91 . Va n Esch . G.J. . an d Kroes . R . (1969) . Brit. J. Cancer23:165. Weber . G. . Singhal . R.L. . Stamm . N.B. . Fisher . E. . an d Mentendick , M.A . (1964) . Adv. Enzyme

Regul. 2:\. Weiss . I.W. . Morgan . K. . an d Phang . J.M . (1972)./ . Biol. Chem. 247:160. Witschi . H. . Kaccw. S. . Tsang . B.K. . an d Williamson . D . (1976) . Chem.-Biol. Interact. 12:19. 

# Lea d an d Hem e Biosynthesi s Michael R. Moore. PeterA. Meredith aad Abraham Goldberg

TABL E O F CONTENT S

I . INTRODUCTIO N - 8 0 II . TH E HEM E BIOSYNTHETI C PATHWA Y8 0 A , Dqlia-aminolevulini c Aci d Synthas e 8 3 B . Oelia-AminolovulMii c Aci d Dehydratas e 8 3 C Uroporphyrinoge n 1 Synthas e an d UroporphyrinogenAitynthetas a 8 3 O . Uroporphyrinoge n Doctvhoxyl-is e 8 4 E CnprotKwphyrinoae n Oxrda M 8 4 F Protopofphyrinoge n Oxidas e 8 4 G . Fnrrochelatas e an d Hem e Oxygenns e 8 4 lit . CONTRO L O F TH E HEM E BIOSYNTHETI C PATHWA Y. ' 8 5 IV . SOURCE S O F LEA D EXPOSUR E 8 8 V . LOCALIZATIO N O F LEA D WITHI N TH E BOD Y VI . TH E EFFECT S O F LEA D O N TH E ENZYME S O F TH E HEM E BIOSY N THETI C PATHWA Y8 9 A . AL A Dehydratas e 8 9 B . Coproporphyrinoga n Oxidas e an d Ferro chelatas e 2 . ._. C . AL A Synthas e an d Hem e Otygenae * 9 1 D . Othe r Effect * 9 2 Vlt . CHANGE S I N TH E PRODUCTIO N AN D EXCRE -

. TIO N O F PATHWA YINTERMEDIATE S DURIN G LEA O EXPOSUR E 9 3 A . Delta - aminotovulintnt t Aci d 9 3 B . Porphobilinoge n 9 S C . Coproporphyri n an d Protoporphyri n 9 5 VIII . TH E DIAGNOSTI C US E O F EXCRETIO N AN D ACTIVIT YPARAMETER S 9 6 A . Delta-aminolevulini c Aci d Concentration * i n Urin e 9 7 B . Bloo d Concentration s ofAL A 9 8 C Urinar y Coproporphyri n 9 9 D . Bloo d Protoporphyri n 10 0 E . Eiyihrocyt e delta - aminolevulini c Aci d Dehydratas e 10 0 F . Inte r compariso n o fIh e Relativ e Valu e o f Hem e Bioiynthatr c P.iramelvr a i n Assesi ' men t o fLea d Exposur e 10 2 G . Hemoglobi n 10 3 IX . CHANGE S I N DRU G METABOLIS M DURIN G LEA D EXPOSUR E 10 3 A . Anima l Studie s 10 4 B . Huma n Studie s 10 8 7 «, , WJlSS , »*.'** , VTT* ••

X . TH E ANEMI A O F LEA D POISONIN G 10 7 XI . A COMPARISO N O F LEA D POISONIN G AN D ACUT E HEPATI C PORPHYRI A 10 9 A . Genera l Consideration s 10 9 B . Th e Irrfluenc a ofOetta-aminotvtlltni e Ael d 11 2 XII . SUMMAR YAN D CONCLUSION S 11 4

I . INTRODUCTIO N Th e effec t o flea d o n hem e biosynthesi s has bee n lon g recognized . Indeed , a grea t similarit y ha

s bee n note d betwee n som e aspects o flea d poisonin g an d th e symptomatolog y o facut e porphyrias . Hematoporphyrinuri a wa s note d h y Stokvi s (1895) . Pcrul z (1910 ) an d Gnetz l (1911 ) i n lead-poisone d rabbits , l.acnne c (1831 ) note d tha i patient s wh o die d oflea d poisonin g showoi l thin nes s o fbloo d an d pallo ru ftissue s o n postmorte m examination . Binnchdij k (cf . Stokvis . IS95 ) firs t note d th e occurrenc e o f exces s porphyri n i n th e urin e o f a patien t wit h lea d intoxicatio n an d i n 192 7 l.cibi g suggested tha i th e ris e i n porphyri n excretio n durin g lea d poisonin g wa s du e l o th e actio n o flea d upo n hemoglobi n synthesi s i n Ih e hon e marrow , (iroctpas s (1932 ) showe d that enhance d urinar y porphyrin s wer e du e principall y t o th e eleva tio n i n coproporphyri n series II I isomer . Mor e recently . Ilaege r (1957 ) foun d excessiv

e urinar y excretio n o f {-aminolevulini c aci d durin g lea d intoxication , l l is cl<:ar fro m these earl y pointer s tha t lead ha s very drmnati c effect s upo n th e biosynthesi s o fheme .

II . TH E HEM E BIOSYNTHETI C PATHWA Y Th e origin s o fhem e biosynthesi s li e i n th e citri c aci d cycl e an d i n norma l C , amin o aci d metabolism . Th e elucidatio n o f th e earl y sleps o f th e hem e biosyntheti c pathwa y wa s th e resul t o f a n elegan t se t o f isotopi c trace r studie s carrie d ou t b y Shcmi n (1955) . I n th e initia l slag e o f th e pathway , succinat e a

s succiny l Co A combine s wit h glycine , unde rth e aegi s o f th e initia l enzym e o r th e pathway , {-aminolevulini c aci d synthase (E C 2,3.1.37 ) an d th e cofacto rpyridoxa l phosphat e t o for m th e firs t ofth e intermediate s o fth e pathway , {-aminolevulini c acid . I n th e nex t stag e o fth e biosyntheti c pathwa y tw o mole s o f{-aminolevulini c aci d condens e l o for m th e monopyr role . porphobilinogen , b y th e actio n o f th e enzym e {-aminolevulini c aci d dehydratas e (porphobilinoge n synthase ) (R C 4,2.1.24) . I n th e next stag e o fbiosynthesis , fou rmole s o f porphobilinoge n condense , cycliz e an d iso mcriz c t o for m th e first o f th e porphyrins , uroporphyrinogen , a s th e thre e isomer . Thi s i s carrie d ou t b y th e concerte d actio n o f tw o enzymes , uroporphyrinoge n I synthas e (porphobilinoge n deaminase ) (E C 4.3.1.8) . an d uroporphyrinoge n cosynthase . I n circumstance s wher e ther e i s a defi - * I T* fSoiH***"**"* ! !«,' ' jT'f \-A : X. 9

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Heur e I . Th e hem e hiossnthcti c pathway . Hem e hiossnlhesi s withi n Ih r cel l start s i n th e mitochondrio n wil h ih e initia l ani l ral e limitin g enzym e AL A synthase . Synthesi s Ihe n passe s int o th e cytoplas m an d rclurn s onc e agai n H i th e mitochondrio n To r His ' final thre e stapes i n Ih e pathway."Contro l o f th e ptlhwa y i s h y negativ e feedbac k h y hem e upo n AL A synthase . Th e enrsme s o f th e pathwa y ar e show n withi n Ih e ope n square s an d th e intermediate s o f Ih e p.ithwa vfolln wth e heav y arrow s t o Ih e final product , heme . Eac h o f ih e intermediates : ma y h e use d i n th e siihw|ticn t productio n n fth e followin g componen t o f Ih e paihsvay . esceplin g th e serie s I porphyrinopens . uroporphyrinoge n I an d coproporphyrinoge n I . whic h ma y no t h e utilize d beyon d th e stag e o f coproporphyrinoge n I an d ar e therefor e obligatoril y excreted . Abbreviations : ALA . iaminolcvulini c acid : PBG . porphobilinogen : COOII . cathoxyl .

ciency o f uroporphyrinoge n cosynthase . series I isome ri s formed . By suc cessiv e decarboxylation s o fth e octacarboxylli c porphyri n ringjlhroug h Ih e hepta . hex a an d penta-carboxyli c porphyrins , the^tetracarboxyli c coproporphyri n i s forme d b y th e reactio n catalyze d -by ^ th e enzym e uroporphyrinoge n decarboxylas e (E C 4.1.1.37) . A t thi s stag e th e reactio n re-enter s th e mitochondrion . whereMCoproporphyrinogen ' 'II I i s decar boxylate d b

y coproporphyrinoge n oxidas e (E C 1.3.3.3 ) to'/or m proto porphyrinoge n IX . whic h i s a dicarboxyli c diviny l porphyri n -Itftji e reduc in g milie u o f th e mitochondrion . pfotoporphyVinoge n IX"*i s oxidize d t o protoporphyri n b y protopb'rphyrnoge n oxidase . Th e final ste p o r th e pathwa y i s th e insertio n Vf-fcrrou s iro n int o protoporphyri n b y ferrochel atas e (E C 4.99.1.1 ) t o for m hem e (Fig . I) . Th e on e othe rcomponen t o fthi s sequence tha t shoul d b e mentione d i s th e first o r th e biodcgradaliv e steps o f heme . I n this , b y splittin g o f th e hem e rin g wit h loss o r iron , hem e oxygenas e (E C 1.14.99.3 ) produce s bilirubi n (Tabl e I) . 

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Commo n namc(s ) i-Amtnolevulinat c synthas e Porphobilinoge n synthas e 4-Aminolcsulinat c dehydratase ) Uroporphyrinoge n I synthase ) Porphobilinoge n deaminase ) Uroporphyrinoge n i

cosynlhctas e Uroporphyrinoge n decarboxylas e Coproporphyrinoge n oxidase ) Coproporphyrinogcnase ) Protoporphyrinoge n oxidas e Ferrochelatase ) Hem e synthetase ) Heme-oxygenas e Abbreviatio n Systemati c nam e ALA. S , Succinyl-CoA : Glycin e C-Succinyltransfcras e ALA. D 5 Aminoevulinat c hydro-lyas c URO. S Porphobilinoge n ammonia-lyas e " r "' " ' URO.Co S •Unclassifie d URO. D ' Uroporphyrinoge n 3 carboxy-lyas e COPRO. O Coproporphyrinogen : Oxyge n oxidoreductas e PROTO. O Unclassifie d FERRO. C Protohcm e fcrro-lyas e HEME. O . Heme : NADPH : Oxyge n oxido-reductas c Enzym e classificatio n nomenclatur e 2.3.1.3 7 4.2.1.2 4 

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porphobilinoge n molecules . Th e actio n o fbot h UR O synthas e an d uroporphyrinoge n M l cosynthas e catalyse s th e hcad-to-hca d condensatio n o f th e porphobilinoge n molecules . Thi s result s i n th e formation o fa n altere d enzyme-boun d dipyrrylmethan e whic h i s specificall y incorporate d int o uroporphyrinoge n II I (Frydma n e t al. , 1976 ; Battersb y an d McDonald , 1976) . D . Uroporphyrinoge n Decarboxylas e Th e final cytoplasmi c ste p o fhem e biosynthesi s involves tin : decarboxyla tio n o fth e octa-carboxyli c uroporphyrinoge n t o th e teti a carbo.xyli c coproporphyrinoge n III . Th e decarboxylatio n catalyze d b y uropor phyrinoge n (URO ) decarboxylas e i s a stepwis e process , th e aceti c aci d group s bein g dccarboxylatc d i n a sequentia l clockwis e fashio n startin g u il h tha t o n rin g I I an d followe d b y thos e o fIh e A , B ant ) ( J ring s (J.-irkso n c t al. , 1976) . Th e specificity o fUR O decarboxylas e is fo rIh e scri-.-- ; II I isome r o furoporphyrinoge n whic h i s decarboxylatc d twice a s rapidl y a

s th e scries I isomer , bu t i t wil l decarboxylat e th e scries I isome rt o pmduc c iroproporphyrinogc n I whic h i s excrete d a s coproporphyri n I (Fig . 2) . K . Coproporphyrinoge n Oxidas e A t thi s stag e i n th e pathwa y th e reactio n scries rc-cntcrs th e mitochondrio n fo rth e conversio n o fcoproporphyrinoge n II I t o protoporphyrinoge n I Xb y th e decarboxylatio n o ftw o propioni c sid e chain s l o viny l groups . Th e enzym e coproporphyrinoge n oxidas e catalyze s th e conversio n o fcopro porphyrinoge n II I t o protoporphyrinoge n IX . Th e mechanis m fo rthi s reactio n i s no

t altogethe rclear , bu t th e evidenc e i s compatibl e wit h thre e closel y relate d mechanisti c alternative s (Akhta re t al. , 1976) . whereby ther e i s a stepwis e decarboxylatio n o fth e propiony l group s t o viny l group s wit h a monoviny l porphyrinoge n existin g a s a n intermediate . F . Protoporphyrinoge n Oxidas e Unti l recently , indirec t evidenc e suggested tha t th e oxidativ e conversio n o f protoporphyrinoge n I Xt o protoporphyri n I Xi n th e reducin g milie u o fth e mitochondrio n wa s controlle d enzymaticall y (Porr a an d Falk . 1964) . Jackso n c

t al . (1974 ) conclusivel y demonstrate d tha t thi s wa s true . Th e enzym e protoporphyrinoge n oxidas e has subsequentl y bee n isolate d fro m bot h microorganism s (Poulso n an d Polglase , I97S ) an d mammalia n tissu e (Poulson , 1976) . G . Ferrochelatas e an d Hem e Oxygenas e Th e fina l ste p i n thi s biosyntheti c pathwa y i s th e insertio n o fferrou s iro n int o protoporphyri n I Xb y ferrochelatas e t o for m Heme . Thi s enzym e i s widel y distribute d i n natur e an d a s a particulat e enzym e i s foun d associate d PORPHYRI N "OUPHYIINOCt N

AC E « 0 [ I liopmpl'y'i n I oqen ) I AcHy l Pmpiony l Act'y t P>opi.-ny l Atriy l Piopiony t 3 ' ' l-eopinny l Acply l Cipropo>pl,y finfngrn) ! M*il,y l Mtihy l " M^>hy l Pmpiony l J Propipny t MetKy l PiO'opo,pl,ytS n S * " Viny l " tigur e 2 . Th e structure o fth e porphyrin s am i porphyrinogens . B y Attachmen t o fth e lipand s sll'nvn il l th e position s A t o I I roiim l th e porphjri n o rporphyrinoge n n'tclcus . eac h o r th e component

s o fth e liiosynlheti c pathwa y o fhem e is shown . Hem e itsel fis forme d h y insertio n offerrou s iro n int o th e cente r ofth e protoporphyri n s ) nucleus . Th e porphyri n is th e osidire d for m ofth e porphyrinogen , whic h i s th e norma l biosyntheti c intermediate .
wit h th e inne rmitochondria l membran e i n animal s (Macka y e t al. . 1969 ) an d boun d t o chromalophorc s i n microorganism s (Neuberge r an d Tait , 1964) . Th e mechanis m o fthi s enzymi c reactio n ha s no t ye t bee n clearl y elu cidated , bu l o fal l th e factor s affectin g ferrochelatas e activit y lipids , an d especiall y phospholipids , see m t o have th e greates t effect (Yoneyam a e t al. , 1969) . Hem e oxygenas e catalyze s th e initia l rate-limitin g ste p o fhem e deg radatio n b y preferentiall y splittin g th e tetrapyrrol c betwee n th e A an d D rings , thu s producin g th e linea rtetrapyrrol c bilirubi n 9 n wit h consequen t los s o firo n an d o fcarbo n monoxide . Thi s reactio n whic h i s microsoma l require s oxyge n an d NADPII . III . CONTRO L O KTH E IIKM E RIOSYNTIIKTI C PATHWA Y I n norma l circumstances , thi s i s a highl y efficien t an d coordinate d pathwa y producin g onl y sufficien t amount s o fth e intermediate s an d ultimatel y o f hem e t o servic e requirement s fo rhemoglobi n an d olhe r hemoprotei n syn i i:, ; ! I. 1

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* Le w Urln . figur e 4 . Distnhutio n o f lea d ssithi n th e body . Th e thre e source s o f lea d intak e ar e show n here . Dietar y intak e wil l resul tin abou t10' J adsorptio n fro m th e gut . Pulmonar vintak e wil l resul tin abou t40- , absorptio n fro m th e lungs . Percutaneou s ahsnrptio n wil l resul tpenc rill y i n anegligibl e intak e throug h th e skin , exceptin g wher e lea d i s in th e form o f a n alks l lea d com poun d whic h ma y h e absorbe d u p t o 1 % o rapplie d material , followin g absorption . La d i t transporte d b y Ih e bloo d l o Ih e sof ttissue s an d t o th e bone . Greater , tha n 90 ^ o f th e tota l bod y lea d burde n wil l h e foun d in bone , wil h relativel y hig h concentration s in live ran d kidncs s Uri nary excretio n account s fo rIh e greate rsourc e o f lea d loss , althoug h som e ma y als o h e e'screte d in th e feces . Re d bloo d corpuscle s show n b y a n ope n circle : Lea d show n hy aclose d triangl e 

### Lea d has man

y divers e biochemical efTccts , all o f whic h ar e o f adeleteriou s nature . N o evidence ha s been presented fora n essential functio n forlea d i n metabolism . Lead has avalenc e shell electroni cstructure o f 6s * 6p ' and nor mally exhibits avalence o f two . Th e vacan t6 p orbital s ma y b e filled by th e formation o f

covalcntbonds ; thi s occur s mostreadily i n biological system s wit h sulphuratom s in proteins . Thes e biochemical effects , therefore , al l probably relat

e ultimatel y t o th e capacity of lead t o combin e wit

h specifi c biochemica l ligamls . Thes e includ e sulfhydry l groups , amin o groups , carboxyl groups , pheno.x y group s and imidazol e residues . I n suc h circum stances , lea d mightb e expecte d t o alte rtertiar y structures o f

biochemical molecule s and , therefore , alterordestroy theirbiochemical function . Con sequently , lead can chang e enzym e activity an d destro y th e structure-func tio n relationship s o f

nuclei cacids . I ti s clcnrfro

m man y studie s Iha

l lead ca n markedly inhibi tmitochon dria l respiration . I

n additio n l o lit

e defectiv e structur

e of theirmembranes , isolate d mitochondri asho wimpaired oxidativ e phosphorylatio n and . ulti mately , impaired mitochondrial respiration . Of the comple

xof enzyme s tha

tsynthesis e succinyl Co A fro m n-ket o glutami cth e dilhio l enzym e lipoamid e dehydrogenas e i s markedl y inhibite d by lead . Thi s inhibitio n i s probably representative ofth e effects o f lead o n man y enzyme s eithe rcontainin g sulfhydry l group s oractivate d by sulfhydryl groups . A . AL A Dehydratas e ____ __ I ti s clearfro

m consideratio n o f

excretio n patterns during lead exposur e tha tspecifi cstage s during the biosyntheti cpathwa y ar e particularly vulnera bl e t o th e effect s oflead . Studie s hav e no wbeen carried outo n mos tenzyme s o rth e pathwa

y an d hav e indicate d both i n anima l an d i n human studie s jus t whic h stage s are mos tsusceptibl e t o change . Th e mos tdiscusse d enzym e i n thi s respec ti s {-aminolevulini caci d dehydratas e which , durin g lea d exposure , ha s clearly been shown t o b e markedly inhibite d (Hernber g and Nikkancn . 1972 ) (Fig . 5). Thi s results i n excessiv e production and excretio n o f 6-aminolevulini cacid . Th e mechanis m of thi s inhibitio n remain s unclear , butiti s associate d wit

h alteration s in th e concentratio n o f free sulfhydryl group s i n biological systems . Th e availabilit y o f concentration s o f

othe r metal s suc

h as zincmay als o contribute , sinc e zinchas been shown t o activate AI. A dehydratas e (Meredit h and Moore . 1978) . Organi clead poisonin g i s rare : thu s ther e arconl y asmal l numbe ro f studie s showin g it

s effects on the hem e biosyntheti cenzymes . Wha tstudie s there ar e sugges tthatth e effects o f organi clead o n thes e enzyme s are simila rl o thos e o f

inorgani clead . Millare tal . (1972 ) showe d thatdiethy l-xir-,^*^rA*v-*rsrwv^-s'^-<:^,r*wr** ^

thesi s withi n th e cell . I n som e circumstances , contro l o fth e pathwa y ca n be s o altere d b

y gros s disturbance s o fth e biosyntheti c sequenc e as t o produc e ver y larg e quantitie s o fredundan t precursors whic h must the n be excreted . L-.:-' l poisonin g i s on e circumstanc e wher e suc h a n effect ca n tak e place . Severa l feature s combin e t o mak e th e hem e biosyntheti c pathwa y easily , an d accuratel y controlled . Th e firs t importan t featur e i s tha t hem e act s b y feedbac k

repressio n an d inhibitio n upo n th e initia l enzym e o fth e pathway , ^-aminolevulini c aci d synthase . Thi s ste p the n becomes th e rate cntrollin g ste p o fIh e pathway . Thi s contro l i s aide d b y th e shor

t half-lif e o t thi s enzyme : i t i s abou t I l o I 'h h ri n mammalia n system s (Moor e an d Goldberg . 1974) . Contro l i s furthe raide d b y membran e permeabilit y considerations , sinc e durin g th e sequence o fth e pathwa y (h e produc t passe s fro m th e mitochondrio n t o th e cytoplas m an d the n bac k int o th e mitochondrio n again . I fat an y stag e i n th e pathwa y oxidatio n docs take plac e o fth e porphyrinoge n l o th e porphyrin , suc h porphyri n must b e excrete d since i t canno t b e furthe rused i n th e biosyntheti c sequence , l-qually , an y deficienc y o fth e enzym e uroporphyrinoge n cosynthas e leadin g t o productio n o fscries I isome rporphyri n wil l requir e tha t suc h porphyri n i s als o excrete d sinc e i t ma y not b e use d beyon d th e slag e o fcoproporphyri n I i n norma l biosyntheti c mechanisms . I t i s clear , therefore , tha t an y circum stance tha t ca n alte rIh e relativ e proportion s o factivit y o fthese enzyme s wil l alte rth e quantit y o fprecurso rproduce d durin g th e progres s o fth e pathway .
IV . SOi;R { F.

S O KLEA D EXPOSUR E Ther e ar e multipl e sources o r lea d exposure withi n th e environment . Mos t o fthes e ar e summarize d i n Figur e 3 an d al l deriv e ultimatel y fro m th e lea d i n th e earth' s crust . Fo rmoder n man . th e greatest exposure take s plac e throug h th e foo d tha t h e eat s an d th e wate rtha t h e drinks, althoug h i n specifi c circumstance s inhalatio n o fai rwhic h i s contaminate d wit h lea d o r uptake o fdust fro m industria l processe s ma y represen t importan t exposure vectors (Fig . 3) .
V . LOCALIZATIO N O F LEA D WITHI N TH E BOD Y Th e actua l sites withi n th e bod y i n whic h lea d i s deposite d ar e a s importan t a featur e as . an d par t o fit s effec t upon , th e hem e biosyntheti c pathway . Althoug h durin g th e absorptiv e processe s lea d is foun d i n exces s i n Ih e blood , followin g absorptio n i t i s rapidl y transferre d to . sof t tissue s an d ulti matel y end s a

s depot lea d i n bone . Withi n th e bon e lea d is relativel y inert , ye t i n exces

s o f95 % o fth e whol e bod y lea d burde n wil l be foun d i n th e bone . I n these circumstances , lea d i s readil y availabl e t o th e marrow . I n Figur e J . Th e vector s o fhuma n lea d exposure

. Th e source s from whic

h ma n ma y b e expose d t o lea d ar e show n h y th e arrows, w-it

h th e heavie rarrow s indicating th

e greate rdegres s o f exposure . Th e principal sourc

e o fhuma n uptak e o flea d i s through foo

d an d wate rconsumption . Absorptio

n ma y als o tak e plac e through th

e ai r breathed , b

y percutaneou

s absorptio

n o f alky l lea d compound

s i n gasoline

s an d through variou

s source s o fcontamination .

consequence th e processe s o fre d cel l growt h an d maturatio n and . o fcourse , formatio n o fhemoglobi n fo rthese cell s wil l be inhibited . I n th e sof t tissues , lea d i s foun d i n greates t concentration s i n th e kidney , th e principa l lea d excretory organ . Lowe rbu t significan t concentration s o flea d ar e als o foun d i n live ran d i n spleen . Th e distributio n o flea d withi n th e bod y i s show n i n Figur e 4 . I t ma y be note d tha t althoug h onl y 10 % o r ingeste d lea d i s absorbed , u p t o 40 % o finhale d lea d wil l be take n i n b y th e pulmonar y route , providin g th e particl e siz e i s sufficientl y small . Percutaneou s absorp tio n o flea d i s unlikel y t o pla y an y importan t rol e i n it s uptak e unless th e lea d exist s i n a lipi d solubl e form , suc h as th e alky l lea d compound s (Fig . 4) . • I I 

## fc : 

## -H» xS&^fY. Y• • r4 tt'.^m>r*a -»/.» • X\—..,*•; • „• ,



## « H

ERYTHROCYT E ALA. D [nmo l ALA/mtn / m l ,.b.. |

2 3 BLOOD Pb(wmol/l | Figur e 5

. Th e relationshi p betwee n bloo d lea d concentration s an d erythrocyt e &-aminnlevu
- ~linic~aci d dehydratase'activity

. A s bloo d lea d concentration s ris e t h h'umans

. s o th e activity~b f erythrocyt e AL Adehydratas e fall s exponentially , wit h th e greates t rat e o fdecreas e bein g see n a l lowe rbloo d lea d concentrations

. Fo rthi s graph , th e mathematica l relationshi p i s eryth
rocyt e ALA. D - 58le- , ""~ " •—' + 0.06

. lea d depresses ALA. D activit y whil e Beatti e et al . (1972) . i n investigation s o n fou rsubjects exposed t o lea d alkyls . demonstrate d a marke d depressio n i n erythrocyt e ALA. D activit y an d a modest elevatio n o rerythrocyt e protoporphyri n (PROTO) . bu t n o elevation s i n urinar y AL A an d COPRO . B . Coproporphyrinoge n Oxidis e an d Ferrochelatas e Th e nex t stag e i n th e pathwa y tha t i s clearl y inhibite d b y lea d i s a t th e leve l o fcoproporphyrinoge n oxidatio n a s show n b y excessiv

e urinar y excretio n o f coproporphyrin . Recen t studie s have show n tha t lea d exposure i n ma n an d animal s result s i n highl y significan t depressio n o factivit y o f coproporphyrinoge n oxidas e withi n th e mitochondrio n (Campbel l e t al. , 1977) . Th e last enzym e ofth e pathway , ferrochelatase . has als o bee n show n l o be inhibite d b y lea d an d ma y contribut e t o th e sideroblasti c types o f anemi a tha t develo p durin g lea d poisonin g (Campbel l e t al. . 1977 ) and , i n addition , t o th e marke d ris e i n protoporphyri n synthesi s (Fig . 6) . C . AL A Synthas e an d Hem e Oxygenas e Th e activit y o ftw o enzyme s i n th e biodegradativ e an d biosyntheti c sequence ar e know n t o b

e elevate d durin g lea d exposure . Recen t anima l experiment s have demonstrate d tha t th e activit y o fhem e oxygenas e i s raise d b y lea d presentmen t (Maine s ani l Kappas , 1976a : Goldber g c t al. . 1978) . Th e ne

t effec t o fdiminishe d rcrrnchelalas c activit y an d raise d hem e oxygenas e activit y i s t o lowe rth e availabilit y o r fre e hem e concentration s withi n hio lugica l system s b

y virtu e o fth e feedbac k contro l o fhem e upo n th e activit y o fth e initia l an d ratc-limilin g enzym e o fth e pathway . Th e resul t i s tha t th e activit y o fthi s enzym e o-nminolevulini c synthase i s raise d durin g lea d exposure (Maxwel l an d Meyer . 1976). presumabl y i n a n attemp t t o com pensate fo rdiminishe d hem e productio n i n thes e circumstances . Th e com binatio n o fincrease d activit y o fAL A synthase an d decrease d activit y o f AI. A dehydratas e ca n accoun t i n kineti c term s lo r th e ver y marke d increas e i n circulatin g an d excrete d AI. A durin g len d exposure (Meredit h e l al. . 1978) . Thes e changes i n hem e biosynthesi s ar e detaile d i n Fig . 7 .

Figur e 6

. Th e effect s o flea d exposure o n coproporphyrinoge n oxidas e nn d ferrochelatase

. Th e activit yo fthes e tw o cn/yme s shows a pronounce d diminutio n o factivit yafte rindustria l lea d exposure . Coproporphyrinoge n oxidase

. COPRO.O ; units , n mo l protoporphyri n utili/ed/ g nrotein/hr ; ferrochelatase , FKRRO.C : units , percentage , uptak e o f"F e int o heme/hr

. '.'-ii? - j'.n2J..*.t; - ;••- !•ffttc to f lea d on •ntyn * activit y

AL U « - (urlM ) 

# II

COPR O * - lurlne l

PR0T 0 « - (blood ) GLTciir e • socciim . Co *
I M A Syntha M 1-MUKOLAEVtrLMI C ACI D (MA )
AIA Dehydratas e PORPHOBILIMOCt

* (PBO) UR

O 1 Syntha M

0R 0 Coaynthaa e UROPORPHYRINOGEN:!i ! (ufo )

r I UR

O Oecarboxyla« « COPROPORPHYRINOGEN'II I (COPRO

)
I COPR

O Oxidat e • PR0T0P0RPHTRJNOGC

N Protoporphyrlnoga n

Oxidat e PROTOPORPHYRI

N I X (FROTO

) ra-rrochalatat a 

## ~l • • HAE M Figur e 7 . Th e efTcct s o f lea d o n hem e biosynthesis . Lea d inhibit s thre e enzyme s o f hem e biosynthesis : thes e ar e ^-aminolevulini caci d dehydratase , coproporphyrinoge n oxidas e an d fer rochelatase . A s aconsequenc e o f decrease d hem e production , ther e i s aris e in th e activit y o f th e initia l ratc-limitin g enzym e ^-aminolevulini caci d synthase . A s afunctio n o f thes e block s i n hem e biosynthesi s an d th e increase d inpu to f AL A int o Ih e biosyntheti cpathway , thre e inter mediate s o f

th e pathwa y ar e produce d i n excess . Th e firs tt i o-aminnlcvulini cacid , whic h i s excrete d i n th e urine : th e secon d i s coproporphyrin . als o excrete d i n th e urin e Anally , protoporphyri n i s produce d an d foun d i n exces s i n th e erythrocytes .


### D . OtherEffects Althoug h thes e are th e principa l points a

twhic h lea d ma y affectthe biosyntheti cpathway , otherfeatures have bee n note d during lead exposure . Uncommonly , porphobilinogen concentration s may'rise : thi s ha s been ascribed t o alteration s i n th e activit

y o f uroporphyrinogen I synthas e (porphobilinoge n deaminase) . Change s i n activit y o f uroporphyrinogen I synthas e have been suggeste d as asecondary contro l pointwithi n th e biosyntheti cpathwa

y (Brodi e e l al. . 1977) . I n circumstance s where th e activit y o f thi s enzym e i s raised , on e woul d no texpectelevate d concentra tion s o f porphyrin precursors . I n lead exposure , however , lowere d activit y o f AI. A dehydratas e i s couple d wit h a n increas e i

n uroporphyrinogen I synthase , whic

h will allo wraised concentration s o f {-aminolevulini caci d bu t will generall y notsho welevate d concentration s o f porphobilinoge n (Brodi e e tal. , 1977) . Intriguingly , recen tstudie s have shown thatthe activit y o f thi s uroporphyrinogen I synthas e inhibite d during in vitro lead exposur e ma y b e protected fro

m the effects of

loa d b y availabilit

y o f pteridine derivative s suc h as pteroyl hexaglutamate (Pipe ran d Tephly . 1974 : Piperan d Va n l.icr , 1177) . Othe rstudie s have shown tha ta thigherconcentration s o f lea d th e decarboxylatio n o f uroporphyrinoge n l o coproporphyrinoge n i s als o inhibited (Krcimer-Birnhaum an d Orinslcin , 1965) . On e enzym e whic h is almos tcertainl y notaffecte d b y increased lead exposure i s uroporphyrinogen cosynthase . as evidence d b y the excretio n o f

scrie s III isome rporphyrin durin g lea d exposure , rathertha

n excretio n o rserie s 1 isome rporphyrins (Fig . 2) .
VII . CHANGE S I N Till- . PRODUCTIO N AN D EXCRETIO N O F PATHWA Y INTERMEDIATE S DURIN G LEA D EXPOSUR E A . Della-aininolciulini e Aci d 

### Studie s in humans o f

the kineti crelationship s between lead exposure , th e activitie s o r6-aminolevulini caci d synthase , .-aminolevulini caci d dehydratas e an d bloo d ALA concentration s hav e show n apositiv e exponential relationshi p of ALA. S an

d bloo d lead (Fig . 8 ) and anegativ e exponential relationshi p o f ALA. D and bloo d lead (Fig . 5) . Th e associatio n between bloo d AL A and ALA. D and thei rassociatio n consequentl y wit h bloo d lead i s i

n th e for m o f apowercurve . Similarly , there i s apowerrela tionshi p between bloo d AI. A and excretio n o f urinar y AL A (Meredith et al. , 1978) . Thes e results hav e demonstrate d thati n lead intoxicatio n there are larg e increase s notonl y i n urinar y concentration s o f

AL A butals o in circu latin g level s o f

ALA . Thi s elevatio n i s broughtaboutby tw o factors : thes e are , firstly, the depressio n o f ALA. D and . secondly , a n elevatio n ofth e rate limitin g enzym e o f

hem e biosynthesi s ALA.S . Depressio n o f ALA. D by lead i s well documented , bu televatio n o f

ALA. S ha s onl y recently been shown i n lea d intoxicatio n i

n man (Takaku etal. . 1973 ; Campbel l c tal. , 1977) . The'relationshi p o f

raised urinar

y AL A an

d erythrocyte ALA. D show s thatth e consideratio n of such arelationshi p give s limite d metaboli c information . Th e absenc e o f astraight-lin e relationshi p between bloo d AL A*.*~.*r-~-.j~.***•*,-.-•..-r«"-; , ,—^-.»*.,<,** i «l'"'r

. •Mv*,r*MF*s«.- - 9 4 Leas t I o.xicit y

" 0 I J I 4 J « Bloo d Lr>.-i d ',)imdl/\\ Figur e H . Th e relationshi p betwee n bloo d lea d concentration s an d leucocyt e i-aminoloulini c aci d synthas e activity . A s bloo d lea d concentration s rise , s o Ih e activil y o f^-aminolevulini c -acid-synlhase-risc s exponentially.-In-lhi s graph.-normat-subject s are-shou- n a s open.circle s and

_ lea d expose d worker s b y close d squares . Th e mathematica l for m o fthi s equatio n i s Leucocyt e ALA. S . 4 » Je •""-"" •+ W.J .
an d urinar y AL A suggest s that , a t highe rbloo d concentration s o fALA , a greate rproportio n i s excrete d int o th e urine . Thi s result s i n a plateau , o f bloo d AL A concentratio n whe n bloo d lea d concentratio n i s i n exces s o f 3 Mmol/1 . A t thi s concentration , urinar y AL A show s a rapi d ris e (Figs . I0 A an d IQB) . Reduce d rena l tubula rreabsorptio n ha s bee n considere d t o be a contributin g facto ri n thi s ris e (Druya n et al. . 1965). However . Crame ran d coworker s (1974 ) showe d tha t AL A clearanc e i s simila rt o th e glomerula r filtration rate , althoug h othe ramin o acid s wer e excrete d i n exces s durin g lea d intoxication . Ther e i s the n a n acceleratin g ris e i n AL A excretio n consisten t wit h reduce d tubula rreabsorption . Thus , belowbloo d AL A concentration s o f4 fimol/l . th e relationship s o fAL A i n urin e an d bloo d ar e linear , l l i s onl y whe n bloo d AL A concentration s ris e above thi s valu e tha t ther e i s a rapi d ris e i n urinar y AL A excretion , possibl y associate d wit h decrease d tubula rreabsorption . Belo wbloo d lea d concentration s o f2 pmol/l . th e relationship s o fAL A i n urin e an d bloo d ar e linear . I t i s onl y Lea d an d Hem e Biosynthesi

s y: > whe n bloo d lea d concentration s ris e above thi s valu e tha t inhibitio n o fAL A dehydratas e and , consequently , hem e biosynthesi s i s sufficientl y grea t t o depres s synthesised fre e heme , induc e AL A synthase activit y an d cause these acceleratin g rises i n synthesize d ALA . Th e interrelationship s o fbloo d lead , leucocyt e ALA.S . erythrocyt e ALA. D an d bloo d AL A ar e o fconsiderabl e metaboli c importance , provide d on e assumes tha

t th e concentration s o fthese parameter s paralle l thos e i n othe rtissues . Thi s i s a reasonabl e assumptio n i fth e individua l i s i n a stead y stat e condition . These relationship * sugges t tha t there ar e "critical " tissu e lea d concentrations , represente d b y a bloo d lea d concentratio n o f approximatel y 2 /imol/ l an d a n erythrocyt e ALA. D activit y o fapprox imatel y 1 8 nmol e AL A utilize d pe rminute/m l re d bloo d cells . Abov e thi s concentratio n an d In-lowthi s activily . th e depressio n i n hem e synthesi s i s sufficien t t o raise th e activit y o fALA.S . th e rate-limitin g enzym e o fhem e biosynthesis , b

y negative feedbac k

(Fig . 9) . II . Porphobilinoge n I t i s not certai n i n wha t manne rlea d poisonin g ma y influenc e th e urinar y excretio n o fPBU . Mos t studies sugges t tha t i t will be normal , althoug h i s som e case

s i t ha s bee n reporte d as raise d (Waldro n an d Stofen , 1974). Th e curren t concept o fcontro l o fhem e biosynthesi s woul d sugges t tha t th e concentratio n shoul d b e norma l i n urin e an d plasm a t o correspon d wit h raise d activit y o furoporphyrinoge n I synthase (Brodi e e t al. , 1977) , althoug h th e activit y o fthi s enzym e has bee n reporte d t o b e inhibite d b y lea d (Pipe r an d Tephly ; 1974) . -• - - . C . Coproporphyri n an d Protoporphyri n Excessiv e urinar y porphyri n excretio n has lon g bee n recognized . Thi s i s cause d principall y b y th e ris e i n coproporphyrin . mainl y as th e serie s II I isomer . Subsequently , i t has bee n show n tha t othe rporphyrin s ma y be detecte d i n increase d amount s i n lea d intoxication . Increase d concentration s o r protoporphyri n ma y b e found . S o als o ca n th e concentratio n o f uroporphyri n rise i n sever e case s o flea d poisoning , wit h associate d increase s i n intermediat e porphyrin s o fth e biosyntheti c pathwa y (Waldro n an d Stofen . 1974) . A notabl e featur e o flea d poisonin g i s th e elevatio n o ferythrocyt e porphyri n levels . Mos t o fthi s i s i n Ih e Tor m o fprotoporphyrin , althoug h erythrocyt e coproporphyri n i s als o raised . Th e elevatio n o r erythrocyt e protoporphyri n ha

s receive d considerabl e attentio n as a bioanalytica l index o flea d exposure . Thi s ha s resulte d i n th e publicatio n o frecommendation s fo rth e us

e o fprotoporphyri n a s a primar y screenin g tes t i n lea d intoxica tio n (Cente rfo rDiseas e Control , 1975) . Protoporphyri n i s foun d bot h i n th e fre e stal e an d a

s a zin c chelate . Bot h o fthes e fluoresc e unde rultraviole t 

## 'I. ! 

# rftsSr"
-

i 

### ':W't<Srs'--if.....- 1 

### v , '•''IE K&4wfer^- irfaswsm-vs.ir/ss*- 9 6 Lea d Toxicit y MOOf

WO O LEUCOCYT E ALA. S ^•no l ALA/K/ B protei n

800 )

t o 2 0 3 0 4 0 ERYTHROCYT E ALA. D •nmo l AL A /mi n /

m l tbc j 5 0 Figur e 9 . Th e relationshi p betwee n erythrocyt e 4-aminotevulini caci d dehydratas e activit y an d leucocyt e ^-aminolevulini caci d synthas e activily . Correspondin g t o Ih e inhibitio n o f ALA. D b y lea d an d th e inductio n o f AL A synthas e b y lead . Ih e interrelationshi p o f thes e tw o enzyme s i s i n ih e form o f

apowe rcurve . I n this graph , norma l subject s ar e show n a s ope n square s ap d lead-expose d subject s a s close d squares . Th e mathematica l for m o f thi s equatio n i s c J t. . . , . „ .. . fleucocvt e ALA. S - 99. 2 Erythrocyt e ALA. P - " ' '



## sD-SSlfc 4

8 3 

# f

ligh tan d resul ti n th e developmen to f fluorocytes . tha tis . erythrocyte s whic h sho wre d fluorescenc e unde rultraviole tligh t(Whitake ran d Vietti . 1958) . VIII . TH E DIAGNOSTI C US E O F EXCRETIO N AN D ACTIVIT Y PARAMETER S I ti s clea rfro m th e variou s feature s o f th e effec to f

lea d upo n Ih e biosyntheti cpathwa y tha tth e change s i n bot h th e activit y o f enzyme s an d th e productio n an d excretio n o f intermediate s o f

th e pathwa y ca n b e use d a s secondar y biologica l indicator s o rlea d intoxication . Althoug h th e earlies t reference s t o th e biologica l effect s o f lea d upo n hem e biosynthesi s referre d t o increase s o f

porphyri n i n th e urine , i ti s no wcertai n fro m consideratio n o f availabl e evidenc e tha tsuc h change s ar e certainl y no tth e mos tsensitiv e o rth e mos teasil y measure d indice s o f exposure , lo rth e purpose s o f thi s section , eac h o f

th e differen tsubstance s an d enzyme s an d thei ruse s wil l b e reviewe d i n turn . Thes e ar e th e concentration s o f o-aminolevulini caci d i n bloo d an d i n urine , (h e concentration s o f coproporphyri n i n urine , th e concentration s o f

protoporphyri n i n th e blood , th e activit y o f o-aminolevu lini caci d dehydratas e i n bloo d and . finally, compariso n o f Ih e relativ e efficienc y o f

eac h o f

these . Als o include d ar e th e effect s o f lea d o n th e concentratio n o f

hemoglobi n i n th e blood , althoug h i ti s certai n tha taver y sever e degre e o f

lea d intoxicatio n mus toccu rfo rchange s i n th e concentra tio n o f hemoglobi n t o h e found . A . Deltn-aminolewdini cAci d Concentration s i n Urin e Th e earl y studie s o f llae<?.ei-Aronse n (I960 ) demonstrate d clearl y tha t measuremen to f

^-aminolevulini caci d i n th e urin e wa s ausefu l diagnosti c tes tfo rlea d exposur e i n lea d workers . Th e method,b y whic h AL A i s measure d i n thes e circumslaucc s ha s alway s bee n th e colorimetri ctech nique , whereb y rS-aminolevuli caci d i s condense d wit h anothe rcompound , usuall y accly l acetone , l o for m a n l-hrlich' s positiv e pyrrole , whic h ma y the n for m apin kcolo rquantitativel y wil h paradimethy l aminohcnzaldchyd e (l-hrlich' s reagent) . Whe n th e concentration s o f urinar y AL A an d bloo d lea d ar e compared , alinea rrelationshi p whic h intercept s th e bloo d lea d axi s a tvalue s abov e th e origi n i s seen . I n th e initia l stage s o f th e graph , change s i n concentratio n ar e s o smal l a s th e b e o f littl e use . However , whe n bloo d

Tabl e 2 . Th e concentration s o f lead , porphyrin s an d porphyri n precursor s in normal an d lead-poisone d human s Norma l subjects Lead-Poisone d

subjects Blood lead fimo\/\

, (Mg/10 0 ml ) j Vrinary AI.A minol/mo l creatinin e (nmol/l ) | Blood

ALA pmol/ l ] Urinary coprrtporphyrin 'umul/rmi l creatinin e : (nmol/l )

Erythrocyte protoporphyrin

(nmol/l ) 0-1.7 5 (0-40 ) 0-2. 5 0-4 3 0.-3. 5 0-12. 5 0-22 0 0-0.7 5 >4. 0

(>80 ) >4. 0 >M> >5. 0 >4 0 >30 0 > I 5 

# u 4$ct#y&.'lV '

Jt< : zK * &;^A:.^.'-'vv * I ta t' * '••'"',V,- : ' ''"•'

- L-Jir^'s^'V

- • ''».-?.:•

. wl?s V ^ 

# fe9 8 Lea d Toxicit y

lea d concentration s excee d norma l limits . I.e., greate rtha n 4 0 <ig % ( 2 /iM) , urinar y AL A concentration s showsom e ris e an d continu e t o d o s o as bloo d concentration s o flea d ris e (Fig . 10) . Norma l value s fo rAL A i n urin e ar e show n i n Tabl e 2 , togethe rwit h th e rang e o fvalue s i n a series o findustriall y exposed workers . B . Rloo d Concentration s o fAI. A Th e concentratio n of/S-aminolevulini c aci d i n th e bloo d may als o be use d a s a n indexo flea d exposure , althoug h th e concentration s likel y l o b e achieve d ar e certainl y no

t as grea t as thos e i n acut e porphyri a i n attack . I n th e norma l person , bloo d concentration s o fri-nminolcvulini c aci d ar c small , bu t followin g industria l lea d exposure they ris e rapidly . Th e mclhoi! - fo r measurin g bloo d AI. A ar e simila rt o thos e fo rurine , except tha t th e bloo d initiall y must b

e treate d wit h iodo-scctamid c t o comple xinterferin g sullhydry l groups , afte rwhic h ALA'wia y be extractci l int o aci d an d



# 3 <

* Bloo d Lea d [uM i Figur e 10

. th e relationshi p betwee n bloo d lea d an d hloo d an d urinar y^-aminolevulini cacid

. A s bloo d lea d concentration s rise , s o th e concentratio n o f th e urinar yexcretio n o f 6-amino
levulini caci d rise s exponentiall y(A)

. Bloo d AL Aconcentration s showa mor e comple xrelationshi p whic h i s probabl yrelate d l o change s i n ih e urinar yeserelio n o f AL A(R)

. Th e mathematica l form s o fIhcse'rclationship*ar e

Urinar yAL A .OSfle*""

—"• * »04 l Bloo d AL A

. J.2e-" » - 4l e " • • 4. 9 ~.vv,„<rf^,a»-*-»vs»v*-i'~;«-

.

Lea d an d Hem e Biosvnlhesi s 9 9

BLOO O A t «

2 « MOO D LEA D (ll* .

measure d b y method s simila rt o thos e use d fo rurin e (Haeger-Aronsen , 1960) . Th e mathematica l associatio n betwee n urinar y AL A an d bloo d AL A take s Ih e for m o fa powe rcurve . Howeve ri n industria l exposure th e rela tionshi p betwee n bloo d AI. A an d bloo d lea d i s a n exponentia l one : hloo d AL A concentratio n tend s t o platea u whe n th e bloo d lea d concentratio n i s i n excess o r 3 pmol/ l (Meredit h et al. , 1978 ) (Fig . I0B) . C . Urinar y Coproporphyri n Urinar y coproporphyri n require s mor e complexquantitatio n technique s inherentl y les

s reliabl e tha n thos e fo r.-aminolevulini c acid : consequently , coproporphyri n i n urin e i s generall y a les s satisfactory metho d o fdefinin g 

### i; 1 

## •« vs ; 

## i-*££w?'f' f f 

## ; 

# &tf£%Y Y



# WWm

T J ro- —^".-Wl-,...^—.."/!^,^.^.;.,.,-,. ,

10 0 Lead Toxicit y excessiv e lea d exposure . Lik e {-aminolevulini c acid , changes i n th e urinar y coproporphyri n excretio n star t t o tak e plac e onl y a t greate rconcentration s o fbloo d lea d tha n thos e foun d i n th e genera l populatio n an d ar e therefor e commonl y used onl y i n industria l lea d exposure analysis . Th e norma l value s fo rurinar y coproporphyri n an d typica l value s foun d i n a grou p o r lea d worker' ; ar c show n i n Tabl e 2 . 

## ,. - I) . Dl'io d Protoporphyri n Th e us e o fbloo d protoporphyri n a s a n indexo flea d exposur e has receive d considerabl e attentio n i n th e pas t fe wyears . F.xaminatio n ofthi s indexhas used classica l extractio n procedure s (Alessi o e t al. , 1976 ) an d Ih e develop men t o l microfluoriiuctri c method s wit h whole , hloo d (Knmholt z c t al. . 1972) . Finally , th e developmen t o fa portabl e front-fac e spcctrnlluorinicte r has th e advantag e o frapidit y o fanalysi s an d minima l preparatio n n l material , l l show

s grea t promis e i n Tututc us e fo rexaminatio n o floa d worker s (Blumhcr g c t al. , 1977) . Measuremen t o fprotoporphyri n i n bloo d as a n indexo r lea d exposure i s limite d b y th e very hig h incidence o firo n deficienc y anemia , whic h wil l als o rais e th e concentratio n o fprotoporphyri n i n blond . Suc h objection s have i n par t bee n answere d b y consideratio n o fth e presenc e an d absence o fth e zin c comple xo fprotoporphyrin , whic h i s mor e coniptonl y foun d i n lea d exposure o flon g standing . I t i s iru e t o say , however , tha t i n an y diseas e o flon g standin g i n whic h protoporphyri n i s raised , ther e ar c increase d concentration s o fthi s zin c chelate . Th e highest concentration s o fprotoporphyrin s o fal l i n th e bloo d wil l b e foun d i n subject s wit h erythropoieti c protoporphyri a i n who m th e primar y defec t i s i n th e activit y o fth e enzym e ferrochelatas e i n th e bon e marrow . Norma l value s fo rbloo d protoporphyri n an d value s commonl y foun d i n lea d worker s ar e show n i n Tabl e 2 . Becaus e th e associatio n betwee n protoporphyri n i n th e bloo d an d bloo d lea d show s a positive exponentia l relationshi p (Fig . II) , thi s metho d i s o flittl e us e i n examinin g environ mental lea d exposure , wher e a very hig h incidenc e o ffals e positive s wil l be foun d (Meredit h ctal. , 1979). F . Erythrocyt e Dclta-aminoletulinl c Aci d Dehydratas e A s wil h al l enzym e measurements , th e activil y arrive d a t ca n vary considerabl y dependin g upo n th e method s used t o quantitat e it . Thi s enzym e ha s benefite d considerabl y fro m th e developmen t o fa simpl e stand ardize d metho d fo rth e liuropea n communit y (Berli n an d Schallcr , 1974) , althoug h othe rmethod s fo rmeasuremen t o factivil y ar e equall y acceptable . Al l depen d ultimatel y upo n th e measuremen t o factivit y aroun d th e p H optimu m o f7. 4 o fth e rate s o fformatio n o fporphobilinoge n fro m 4 - aminolevulini c acid , usuall y i n a phosphate buffer . Substances suc h as 4 BLOO D PROTOPORPHYRI N |>imo l EPP/1' | Lead an d Hem e Biosynthesi s 10 1

.«.« •. ° . 1 *
0 1 2 3 4 S

BLOO D P b (iimolfl ) Flror e II . Th e relationshi p betwee n bloo d lea d concentration s an d bio,* ) protoporphyri n concentrations . Bloo d protoporphyri n concentration s ris e exponentially , as bloo d lea d concentration s rise . Th e mathematica l relationshi p i s Bloo d protoporphyri n - n.l4e ,n, ~"'" " *0.4 S alcoho l (Moor e e

t al. . 1971 ) o rzin c (Meredit h an d Moore . 1978 ) ca n interfer e wit h th e activit y o fthi s enzyme , bu t i n genera l lea d ha s th e greates t effec t o r al l upo n th e activit y o fthi s enzyme . Th e associatio n betwee n th e activit y o fth e enzym e an d bloo d lea d concentration s has bee n show n man y time s t o hav e a negativ e exponentia l relationshi p (Fig . 5) . Thi s mean s tha t measuremen t o fth e activit y o fth e enzym e i s a particularl y goo d biologica l indexo r lea d exposure at environmenta l concentrations , i.e.. belo w2 MM -— , .*-. —-.- , :JV > v - - • • -^^.-s : - , .»**,i^W'" » r.*.-»vs* - T . i-v- ^



### F . Intercomparison o f

th e Relativ e Valu e o f Hem e Biosyntheti cParameters in Assessmento f Lea d Exposure I ti s well establishe d thatall th e hem e biosyntheti cparameters w e have considere d ar e highly significantl y correlate d wit h bloo d lea

d levels . A hig

h coefficientdoe s not , however , necessaril y imply ahig h predictive validity . Such validity ca n onl y b e establishe d b y th e determinatio n o f th e exten tt o whic h subjects are correctl y classifie d a s t o thei rbloo d lead level s on th e basi s o f th e heme biosyntheti cparameterunderconsideration . A n evalua tio n of thi s typ e has recently bee n carried ou

t(Meredith . Moore , an d Goldberg 1979 ) on the basi s o f fals e positiv e and fals e negativ e result s a l tw o bloo d lead levels . Th e tw o bloo d lead level s selecte d were 1.7 5 fimol/ l and 3. 0 fitnol/l , the firs

tbein g th e no

wgenerall y accepted desirabl e upperlimi tfor subject s wit h n o known lead exposur e and the secon d being representaliv e o f ahloo d lead valu e thatcoul d b e encountere d i n industria

l lea d exposure . -Fo

r each o f thes e two bloo d lead values , tw o value s each wer e considered fo

ruri nar y an d bloo d ALA , urinar

y COPRO , bloo d protoporphyrin an d eryth rocyte ALA.D . Thes e values were selecte d arbitrarily a s belli}' , thos e must ni.cl y l o resul

ti n hig h predictive validity . Predictive validit y was assesse d i

n term s o f percentag e o f

fals e negatives , thatis , afailure t o detectsubjects wit h raised bloo d lead levels , and percentage o f fals e positives , tha

tis . afals e pre dictio n o f raised bloo d lead levels . Results o f thi s analysi s i

n agrou p o f

sub ject s wit h known industria

l lead exposure and subject s wit h n o know n excessiv e lead exposur e arcshow n i n Tabi ci. I

ti s clearfro

m thi s table tha

t Tabl e 3 . Th e predictivevalidit

y o f hem e hjosynlheticparanicter

s in.th e assessment. , o f elevate d bloo d lea d concentration

s

Blood AL A (<imol/l ) Urinar y AL A (mmol/mo l creatinine ) Urinar y coproporphyri n (iimol/mo l creatinine ) Erythrocyte ALA. D (nmo l ALA/min/m l rb c Blood protoporphyri n («moll-.PP/l ) > J 5 >J. O >2. 5 >2. 0 > 12. 5 > in. o <2 0 <2 5 >I. O >0.7 5 Bloo d lea d Gimol/I )

Environmenta l OI.75 ) False positiv e Cr) 5. 4 18. 6 4. 8 11. 4 10. 2 1 5 6 3. 7 6. 3 6 9 20. 6 False

negativ e 

## r o 9. 0 6. 0 10. 2 5. 4 7. 2 3. 0 7. 4 0. 5 11. 6 4. 2 >4. 5

>4. 0 >5. 0 >4. 0 >22. 5 > 17.5 " < I 0 <I2. 5 >2. 0 >l. 5 Industria l

O3.0 ) False positiv e C O 1. 2 6 6 1.2 4. 2 J O 11. 4 1.6 9: 0 2. 6 9. 0 False

negativ e 

## ('" ) 4.X 1. 8 .1.6 J.O 7. : 3. 6 4. 8 O S 9. 0 4. 2 

### onl y erythrocyte ALA. D offers agoo d inde x o f environmental lea d exposure : tha tis . when the numbero f raise negativ e result

s i s reduced t o a

n acceptabl e leve l {i.e., les s than 5%) by reduction o rth e threshol d value , th e numbero f fals e positive s remains a ta n acceptabl e level (i.e. , les s than 10%) . A tth e higherbloo d lea

d level i ti s apparen

tthat, wit

h th e exceptio n o f

urinar y COPRO . al l the parameters ar e acceptabl e screenin g test s o n th e basi s o rpre dictiv e validity . Thus , forthe screenin g of abroa d population . Ih

e activity o f

eryth rocyte ALA. D i s ahighly sensitiv e inde x of bot h environmental and industria l lead exposure . However , due t o th e relative rapidit y an d sim plicit y o f bloo d protoporphyrin determinatio n usin g th e "hcmatofluo rimeter, " i

ti s probably tha

tthe latte rtesti s mor

e expedientTo

rth e screen in n of a n industrially expose d group o f workers . G . Hemoglobin I ti s only in clearcases o f lead poisonin g tha

themoglobi n level s startt o rail . I n general , this will onl y happe n whe n bloo d lea

d level s ar

ci n exces s o r4 jiMYs O jtg/10 0 ml) . A s show n before . AI. A synthas e activity i s inversely related to hemoglobi n an d hemoprotcin concentration s during lea d exposur e (Campbell etal'., 1977 ; Goldberg e tal. . 1978) ; otherstudie s have shown a simila rassociatio n wit

h bloo d protoporphyrin level s (Meredith c tal. . 1979) . A rise i n bloo d protoporphyrin lc\el s will reflec tdepressio n o rhem e produc tio n more accurately tha n wil l depressio n o rerythrocyte AL A dehydratase . Thi s is consistentwit

h th e previous suggestio n tha tthere are critical tissu e lead concentration s o

T about2 |i M (4O»rg/IO 0 ml) : Thi s correspond s to ~ erythrocyte protoporphyrin level s o rabout0.7 5 m M an d erythrocyte AL A dehydratas e activit

y o r2 0 nmol AL A utilized/min/m l re d bloo d cells . A t thi s concentratio n o rlead , the inhibitio n orheme synthesi s i s sufficientl y grea tto depres s th e synthesize d free hem e and therefore hemoglobi n an d thu s t o induce AL A synthas e activity .
IX. CHANGE

S I N DRU G METABOLIS

M Dl'RING LEA

D EXPOSUR

E A large proportio n o rhem e synthesize d i n th e liveracts a s th e prostheti c group o rth e hemoprotci n cytochrom e P-450 . This hemoprolei n i s th e ter mina l oxidas e o rth e microsomal mixe d fitnetion oxidas e syste m thati s involved i

n the metabolis m o rmany drugs . The possibility thatdru g metabolis m migh

tb e altered i

n lead intoxicatio n due l o depresse d synthesi s o rmicrosomal cytochrom e P-45 0 has bee n considered i

n bot h ma n and ani mals . I I • • '*fJ

'rti^i•• • , v



# 'Ml '• r» f >**.•:>.-.»»:-•«

. 1.»?.ss*

—.-••- ir- —

A

. Anima l Studie s Initia l studie s i n animal s produce d contradictor yresults

. Th e wor k o l Ribeir o (1970 ) o n th e effect s o farsenic , beryllium , lea d an d mercur yo n mous e live rdrug-metabolizin g enzyme s indicate d tha t pretreatmen t o rmic e wit h lea d nitrat e t o giv e live rlevel s o r10' ' an d 10" * M Taile d t o alte r hexobarhita l sleepin g time s o rcertai n microsoma l enzym e activitie s tha t wer e measure d in

vitro. Whe n lea d wa s adde d t o contro l microsome s a t concentration s betwee n 1 0 ' an d 10" ' M , n o inhibitio n o rhexobarhita l oxi
das e wa s noted

. I n contrast , th e result s o fa numbe ro finvestigator s (Alvare s e t al.

. 1972 : Scopp a e t al. , 1973 : Lga n an d Cornish

. 1973 : Cho w an d Cornish

. 1978 ) sugges t tha t lea d ha s a significan t effec t o n microsoma l dru g metabolism

. Alvare s e t al

. (1972 ) foun d a 4 0 t o 50 % decreas e i n metabolis m b yhydroxylatio n an d dcmcthylatio n an d a significan t decreas e i n cytochrom e P-45 0 level s an d hc.xoharbita l sleepin g time s 2 4 hour s afte r' th e intravenou s injectio n o flead chloride

. Fro m th e result s o fthei rstudies , th e author s considere d tha t lead exert s it s effec t o n dru g metabolis m vi a a n inhibitio n o rhem e synthesis

. Scnpp a e t al

. (1973 ) attempte d t o verif ythi s hypothesi s h ycorrelatin g th e inhibitio n o fhloo d an d live rAL Adehydratas e b ylea d wit h dru g metabolism

. The ywer e abl e t o demonstrat e a n associatio n betwee n lowere d activit yo fthi s enzym e an d depressio n i n cytochrom e P-45 0 level s an d a n assoc i itc d depressio n af in vitro activitie s o rIh e microsoma l mixe d functio n oxidas e system

. Thes e studie s di d not , however , provid e unequivoca l evidenc e o fth e existenc e o ra relationshi p betwee n depresse d hem e synthesi s an d altere d dru g metabolism

. Goldber g e t al

. (1978 ) provide d fartherevidenc e tha t lea d ha s a profoun d effec t upo n th e microsoma l mixe d functio n oxidas e system

. A n increase d dos e o flead t o rat s resulte d i n a progressiv e decreas e i n cytochrom e P-45 0 conten t an d a progressiv e decreas e i n th e activitie s o r anilin e hydroxylas e an d aminopyrin e demelhylase

. A s ha d bee n note d pre viousl y(Maxwel l an d Meyer , 1976)

. ther e wa s a n increase i n th e activit yo T th e rate-limitin g enzym e o rhem e biosynthesis

. AL Asynthase , associate d wit h th e impairmen t o fth e mixe d functio n oxidas e system

. A n invers e rela
tionshi p betwee n AL Asynthas e an d cytochrom e P-45 0 conten t was " observed : thi s i s illustrate d i n Figur e 12

. Thi s invers e relationshi p provide s fartherevidenc e o rth e contro l mechanis m whereb ya decreas e i n th e regula
tor ypoo l o rheme , a s assesse d b yth e microsoma l conten t o fth e hemopro
tci n cytochrom e P-450

. result s i n a n increase i n th e activit yo rAL A synthas e b ya negativ e Tecdbac k mechanism

. Fro m in vitro studie s (Scopp a e t al. , 1973 : Goldber g e t al. , 1978) , i t i s eviden t tha t lea d ha s n o direc t marke d effec t o n th e microsoma l mixe d func
tio n oxidas e system

. I t i s apparen t tha t th e depletio n o fcytochrom e P-45 0 125 r



## to o 

## I 

# I

•s rs 0 - 8 07 S

4 ) 

### E O50 r 5 | U il 

## 2 ? ? . I 0-2 5 o — n l i t i i ,

0 5 0 10 0 15 0 20 0

ALA. S (nmol e At- A forme d / 9.homogenat e protein/rs ) 2 5 0

Figur e 12 . Th e change

s i n hepati c microsoma l cytochrom e P-45 0 an d hepati c AI. A synthas e i n lead-lreale d rats . I n thi s graph , th e lead-trcalc d animal , :ir c show n a s close d square s an d th e untreate d animal s are shoss n a s close d circles . As lead concentratio n rises , s o Ih e activity o l AI. A synthas

e rises ; concurrently , ther e is a n exponentia l decreas e i n Ih e concentratio n o f hepati c microsoma l cytochrom e P-450 . Th e mathematica l for m o fthi s relationshi p is Cytochrom

e P-45 0 - •- 0.091 1

i n lea d intoxicatio n i n animal s ca n b e mediate d throug h tw o possibl e mechanisms : th e firs t i s associate d wit h a reductio n i n th e activit yo fth e intermediat e enzyme s o rhem e biosynthesi s AL Adehydratase

. COPR O oxi
das e an d ferrochelatas e synthesi s (Goldber g e t al.

. 1978)

. Th e secon d possi
bl e mechanis m i s associate d wit h a n increase i n hem e degradatio n du e t o

. increase d activit yo fth e hem e degradativ e enzym e hem e oxygenas e (Maine s an d Kappas , 1976b ; Goldber g ct al. , 1978)

. Thi s latte rmechanis m conflict s wit h a previou s mode l suggestin g tha i AL Asynthas e an d hem e oxygenas e ar e interrelate d wit h respec t t o thei rregulatio n b yhem e Bissel l an d Ham
make r(1976)

. The yfurthe rpredicte d tha t thes e enzym e activitie s var y reciprocally

. Thi s theor yha s experimenta l suppor t fro m th e studie s o f Schacte r(1975)

. Fro m thi s theory , on e woul d expec t depletio n ofth e regula
tor yhem e pool , whic h woul d activat e ALA. S bu t depres s hem e oxygenase . Experimenta l evidenc e doc s no t suppor t thi s theory

. Lea d pretreatmen t o fr v , ', ' ..;•*«**. ...M—I—•' * •••^*- *

il.l d I'.A.vll V 

### rats results i

n inhibitio n o rhepati chem e synthesi s and depletio n o rthe regu lator y hem e pool . Thu

s there i s no tonl y a

n associate d increas e i

n th e activity o rAL A synthase , bu tals o aris e in th e activity o rheme oxygenase . Thes e findings are , however , consisten twit h th e theor

y postulated by Maine s an

d Kappas (1976c ) thatth e mod e o f actio n o f

metal s i n inducing hem e oxygenas e i s base d o n arepresso rcomponen ti n the regulator

y mechanis m o rheme oxygenase . They suggeste d tha tthi s repressorha

s a n -S H activ e constituent , th e oxidation-reductio n capacity o l whic h i s necessary forcontrollin g hem e oxygenas e production . I f the oxidation reductio n cycl e o f

Ih e -S M group s o f

thi s componen tar

e blocked , a s would occu raftertreatmentwilh lead , whic h ha s ahig h affinit y for-S H groups , the regulator

y function o f

thi s cellula rconstituen ton heme oxygenas e would be lost . Th e system woul d the n function withoutregressive regulation , lead ing t o a n exaggerate d synthesi s o rth e enzyme . Thus , the impairmento rth e mixed function oxidase s demonstrate d i n lead treated animal s has clearl y been.'shown t o b e mediated throur. h a decreas e i

n hem e biosynthesi s un

d a n increas e i

n heme degradation . As yet . i l i s notpossibl e to conclud e whic h o f thes e two mechanism s i s th e more importanti

n the reductio n o rmicrosomal P-45 0 content ; bot h mechanism s arcpossibl e an

d the y are notmutually exclusive . ft . Huma n Studie s Th e attentio n focused on the effects o rlead o n in vitro drug metabolism i n animal s has resulted i

n anumbero rinvestigation s into th e effects o rlea d exposur e i

n ma n on in vivo drug metabolis m rates . A preliminar y study was carried ou

tby Alvare s e

tal . (1973 ) in tw o children wh o showed clinical and biochemical manirestation s o racute lead poisoning . A s ameasure o rin vivo drug metabolis m rates , phenazon e (antipyrine ) half-live s wer e determined bot h before an

d afterchelatio n therapy . Th e determinatio n of phenazone half-lire i n ma n offers a

n inde xo f drug metabolizin g capacit y becaus e a

n oral dos e i s completel y absorbed and i s metabolize d exclusivel y b y th e hepati ccytochrom e P-45 0 dependentmicrosomal mixe d runction oxidas e system . Th e phenazon e hall-live s i n th e tw o subjects wer

e found t o be sig nificantly longerlha

n i n agroup o rnormal children , whil e chelatin g therapy led t o areduction i

n half-lire i

n bot h subjects . Th e sam e authors (Alvare s e t al. . 1976 ) were , however , unabl e l o demonstrate astatisticall y sigrvficant chang e i n the rat e o f eliminatio n o rphenazon e from (he plasmao reigh t industrially expose d lead workers following chelatio n therapy . The y did note tha ti n seven o f

the eightsubjects ther

e wa s areduction i

n phenazone half-lif e followin g chelatio n therapy . Subsequentstudie s hav

e demonstrate d thati

n acute lead intoxicatio n i n man there is adepressio n in th e rat e o rdrug metabolis m (Meredit h e tal. , 1977 ; Fischbcin e tal. . 1977) . I

n th e firs to rthes e two studie s (Meredith et al. . 1977) , plasmaphenazone eliminatio n rate s wer e determined in agrou p o r1 0 demolitio n workers admitte d t o hospital wit h clinical an

d biochemical symptom s o rexcessiv e lead exposur e before , immediately afterand agai n at leas t1 2 week s afte

rth e cessatio n o f

chelatio n therapy . A tth e sam e time , erythrocyte AL

A dehydratas e activity , bloo d lea d level s an

d hemoglobi n level s wer

e determined . Chelatio n therapy wa s foun d t o b e associate d wit

h a highly significant( p < 0.005 ; paire d t analysis ) decreas e i

n phenazone hair lir e an d a n increase i

n clearanc e bni

h immediately aftertherapy an d agai n whe n measured a

tleas t\2 weeks alte rchelatio n therapy (Fig . 13) . Th e change s i n phenazone eliminatio n rate s following chelatio n therapy were als o associate d wit

h decrease d bloo d lea

d level s an

d increase s i

n bot h hemo globi n level s an

d erythrocyte AL A dehydratas e activity . Th e increas e i n phenazone eliminatio n rate s i n (li e subjects following chelatio n therapy wa s significantl y correlated wit h th e fal l i n bloo d lea

d level s ( p < 0.005) . I n asimilarstud

y on five subject

s wit h biochemical manifestation s o f chroni clead intoxication . Fischbcin e tal . (1977 ) noted aslightbu tstatis ticall y significan tdecreas e in phenazon e half-lif o following chelatio n therapy . However , (he rats- o reliminatio n o rphenylbutazone wa s unaffecte d b y chelatio n therapy . I ti s apparen

ttha tIhe acute administratio n o rlead t o rats i s associate d wilh decrease s i

n cytochrom e P-45 0 contenl o rhepati cmicrosomes . Th e increas e i

n in vivo drug metabolis m rale s measured following chelatio n therapy o rlead-intoxicate d huma n subject s (Meredit

h el al. . 1977 : Fischbci n e tal. , 1977) i s probabl

y due t o depresse d level s o rth e hepati cmicrosomal hemoproteir

i cytochrom e l' ; 

### 450. "
X. TH E ANEMI

A O F LEA D POISONIN

G Thre e mechanism s ar

e involve d in th e developmento ranemi aduring lead poisoning . Firstly , there i s depressio n o rheme biosynthesis ; secondly , there i s disturbanc e orglobi n synthesis ; and . finally, ther e i s hemolysi s o rre

d bloo d cells . Th e developmento rsuch anemi awill occu ra ttwo times , durin g re d bloo d cel l formation and during re d bloo d cel l destruction . Sinc e lead i s preforcntially deposited into bon e (Fig . 4) , i ti s clea rtha

tduring re

d cel l formation i

n the bone marrowthe concentration s o rlead i n th e marrowwil l b e ver y muc h greatertha

n tha ti n th e bloo d (Albaharry . 1972) . I n thes e cir cumstances , the developin g cell s within Ih e bon e marrowwill be particularl y affecte d b

y lead . Therefore on e woul d expectdcrcctiv e erythropoiesi s (Lando we

tal. , 1973) . Ilcr

ketal . (1970 ) suggeste d thaterythropoiesi s i s completel y effective during lea d intoxicatio n i

n ahuma

n adultwit

h lea d poisoning . However , Dag g e tal . (1965) , i

n astud y o f tw o patients wit

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### pre EDT A immediatel y

post-EDT

A > 1 2 week s post-EDTA Figur e 13 . Th e efTcct s o f chelatio n therap y o n phenazon e half-lif e in lead-poisone d workers . Th e rat e o f

phenazon e metabolis m wa s examine d i n 1 0 lea d worker s a tth e lim e o l lea d poison ing , immediatel y afte rethylen e diamin o tetra-aceti caci d (EDTA ) chelatio n therap y an d mor e tha n 1 2 week s afte rchelatio n Iherapy . I n al l cases , th e ral e o f phenazon e metabolis m ros e followin g chelatio n o f

lea d fro m th e system s o f thes e lead-poisone d subjects , a s evidence d b y a decreas e i n ih e phenazon e half-life . Thi s coul d no tb e attribute d l o existin g concentration s o f EDTA . 

### poisonin g before an

d aftertreatmentwit

h penicillamine , showed tha tth e anemi ao f

lea d poisonin g i s due partly t o hemolysi s an d partl y t o inhibitio n o riron utilization . Lea d affects protein synthesis : specifically , i twil l inhibi

tth e synthesi s o rglobin . I

tinterferes wit

h amin o aci d incorporatio n and . whe n reticu locyte s are incubate d wit h lead , on e finds disaggregatio n o f polyribosomes . In vitro experiments hav e shown that , in additio n t o thi s decreas e in globi n synthesis , the rate o rproductio n o ralph aan d betachain s becom e outo r phase , wit

h suppressio n o ralph achai n productio n bein g mor e marked than thato rthe bet

achains . In vivo, there appears t o b e som e compensatio n To r thi s t o restore th e balanc e o rsynthesis : th e significanc e o rthi s finding i s in som e doub t(Whit e and Harvey . 1972) . Th e final effectthatlead may hav e o n th e re d cel l i s it s effecton the re d cel l membrane . Th e norma l lif e spa n o r th e re d cell i s about12

0 da\s . I n hemolyti canemia , th e mean lif e spa n o r thes e cell s measured by radioisotop e technique s i s reduced (Grigg s and Har ris , 1958 ; Waldron , 1966) . Thi s shortenin g o rth e re d cel l lif e spa n i s probably due t o directtoxi ceffectupon th e cell membrane . Lea d interfere s wit

h th e Na'-K * dependentATPas cactivity in th e re d cell (Hasan e

tal. , 1967) : as aconsequence , re d cell s incubated wit

h high concentration s of

lead lea klarge quantitie s o rK' . Thi s los s is irreversibl e and i s thoughtt o b e cause d b y interference wit

h thi s energy-requirin g pump . A tth e sam e time , there i s littl e change i n the inpu

to rNa ' into th e cell ; i

n consequenc e there i s aris e i n Na ' concentratio n i

n lead poisone d eryth rocytes , associate d wit

h potassium-mcdialc d shrinkage o rthe cell . Thi s K ' leakag e almos tcertainl y lead s to a n increased mechanical fragility o rth e re d bloo d cel l an

d consequen tlysis . Th e importanc e 6 ranemi ain lea d poisonin g i s readily seen in th e incidenc e o rth e variou s features orlea d poisonin g shown i

n Figure 1-1

, wher e about90 % o rlead-poisone d workers were found t o hav e som e degre e oranemia . A s th e degre e o rlead poisonin g increases , th e hemoglobi n concentratio n i n the bloo d decreases . A tth e sam e time , ther

e ar e rise s in th e activit

y o r.-aminolevulini caci d synthase , pre sumabl y i

n a n attemp tl o compensate forth e decrease d concentration s o r free heme withi n th e cell . Th e typ e of anemi afound in thes e worker s i s usually anormocytic . normochromi canemia . I n affecte d children , i

tmay becom e macrocyti cand hypochromi canemia . XI . A COMPARISO

N O F LEA D POISONIN

G AN D ACUT E HEPATIC PORPHYRI A A . Genera

l Consideration s Although acut e porphyri aan d lea d poisonin g are condition s o f different etiology , on e bein g geneticall y determine d and th e secon d acquired , i l i s o r interes tt o compar e th e similaritie s o rthei rclinical feature s and o f th e ex cretio n patterns o rporphyrin precursors an d porphyrins . Thes e similaritie s often reflec

tsimilaritie s in th e abnormalitie s o renzym e activity an d porphyrin, metabolis m (Dag g e tal. , 1965 : Gibso n and Goldberg , 1970 ; Campbell etal. , 1977 ; Dos s an d Tiepermann . 1977) . Th e features o f acutelU2-fQlifiBUf i Arun IMTHWITiiai^csrniju a

sfsso* , ino-ii t

bl»'.*Qt a
futlnlAu.cidiAH i liqur e 14 . Th e incidenc e ofth e typica l features o flead poisnninit an d o r acut e intermitten t p'rphvria . Th e incidenc e ofth e effec

t o flead poisonin g i n 1 0 industriall y expose d subjects , al l o fssho m ha d hlood lead concentration s excecdins ; 4 wmol/l . i s demonstrated i n thi s histogram , liifttnc r wil h th e simii-f r incidenc e o ffeatures o facut

e intermitten t porphyri a i n 5 0 subject s iillt ) thi s disease . Sorti e features ar * (Se n onl y i n lead poisoning , such a s hyperuriomia . metalli c tjst c and . i n some case s wher e poo rdenta l hygu-nc

e co-exist

s wil h lead puisonine . ir e gingiva l blu e line . porphyria , whic h ar e abdomina l pain , constipation ; vomiting , paralysi s o r paresis , ar e simila rt o thos e foun d i n lea d intoxicatio n and . indeed , th e Inci dence o fthese i n bot h condition s i s als o simila r(Fig . 14) . I n lea d poisoning , th e paralysi s o rparesi s i s entirel y o fth e motorneuro n type , whil e i n acut e intermitten t porphyri a 10 % o r th e subject s sho wuppe rmoto rncur'or i involvemen t an d organi c brai n disorde r(Goldberg . 1968) . In . term s o r excretio n o r eithe rporphyrin s o rthei rprecursors , th e overal l patter n found i n lea d poisonin g i s i n n o wa y identica l t o an y o r th e porphyrias , althoug h som e similarities exist . I n al l acut e porphyria s there i s excessiv e excretio n o r {-aminolevulini c acid ; th e sam e i s found i n lea d poisoning . I n term s o r porphyri n excretion , non e o r th e porphyria s produc e a patter n simila rt o lea d poisoning : exception s ar e Ih e urinar y excretio n o r coproporphyrin , whic h i s found i n hereditar y coproporphyria , an d th e excessiv e productio n o r protoporphyri n see n i n varicgal c porphyri a an d erythropoieti c protoporphyria . A compariso n o r th e pattern s o fchang e o f enzym e activit y i n lea d poisonin g an d i n acut e intermitten t porphyri a an d hereditar y coproporphyri a show s th e reaso n fo rthese difference s (Fig . 15) . I n acut e intermitten t porphyria , ther e i s depressio n o r porphobilinoge n deaminas e activit y leadin g t o increase d activit y o r AL A synthase , whil e i n hereditar y coproporphyri a ther e i s depressio n o r coproporphyrinoge n oxi dase activit y wit h subsequen t increases i n th e activit y o r AL A synthase . I n lea d poisoning , thre e enzyme s ar e clearl y inhibite d (AL A dehydratase , coproporphyrinoge n oxidas e an d ferrochelatase) wit h consequen t elevatio n i n 6-aminolevulini c aci d synthase . Ther e i s thu s on e feature commo n t o eac h o fthese porphyria s an d lea d poisoning ; tha i i s a rise i n th e activit y o fAI. A synthase , th e onl y othe rfeatur e o r tw o o fIh e acul c porphyria s tha t migh t b e foun d i n lea d poisonin g i s a porphyrin-mediate d sola rphotosensitizalio n of th e skin . I n variegat e porphyria , increase d protoporphyri n lead s to photosensitivity ; i n a propor tio n o fcase

s wit h hereditar y coproporphyria , a photosensitive ski n eruptio n ma y h e found , althoug h thi s is neve rth e case i n acnl c intermitten t porphyria . Pholoscnsitizatio n i n lea d poisonin g i s uncommon , bu t i t has

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••| INCREAS E o o DECREAS E »B ^ S| > ) I N ENZYM E Activit y 0 - N O CHANG E I

N ENZYH E ACTIVITY,KIT H RESPEC T T O NOIWA L VALUE S ALA. S -IAMINOLAEVULINI C ACI DSYNTHAS E ALA. D-HAMINOLAEVULINI C ACI DDEHYDRATAS E URO. S - UROPORPHYRINOGE N 1 SYNTHAS E URO. D- UROPORPHYRINOGE N DECARBOXYLAS E COPRO. 0 - COPROPORPHYRINOGE N OXIDAS E FERRO. C - rtRROCHtLATASE Kipir e 15 . A compariso n o fth e change s i n enzym e activity i n lea d poisoning , acut

e intermit ten t purphyri a an d hereditar y coproporphyria . Althoug h ther e ar e man y similaritie s i n Ih e excretio n o fintermediates o

fIh e heme hinsvnlheti e pathwa y i n lea d poisonin g an d thes e two acut

e porphyrias , the onl y trul y commo n poin t t o al l is Ih e ris e i n activity o f4-aminnlcvulini c aci d synthase . There are als o similaritie s i n depressio n ofcoproporphyrinoge n oxidase i n bot h lea d poisonin g an d hereditar y coproporphyria . 1. 1

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bee n reporte d (Alla n et al. , 1975) . Variou s reason s hav e bee n advance d for th e differenc e i n photosensitizin g lesion s i n lea d poisonin g an d th e cutaneou s porphyrias , suc h as th e presence o fzin c chelate s o r proto porphyri n i n lea d poisonin g (Lamol a e t al. , 1975 ) an d altere d plasm a bind in g o l porphyrin s (Piomell i et al. . 1975) . I t i s possibl e tha t depressio n o r enzym e activit y a t a n earlie rstag e i n hem e biosynthesi s i n lea d poisonin g tha n i n hereditar y coproporphyri a mean s tha t lesse r quantitie s o r substrat e ar c bein g metabolize d b y th e depresse d coproporphyrinoge n oxidase . I n consequence , lesse rquantitie s o r coproporphyri n ar e abl e to buil d u p i n th e circulation . Th e defect ma y als o li e i n oxidation-reductio n factors , since withi n th e bod y Ih e porphyri n normall y exists as th e reduce d porphyrinoge n whic h i s onl y photoactiv e whe n oxidize d t o th e porphyri n for m (l-ig . 2) . Thi s viewi s supporte d b

y th e feature o r altere d termina l oxidatio n durin g lea d exposure . ', . Th e live ri s th e mai n sit e o r disturbanc e o r porphyri n metabolis m i n acute porphyria . Whe n ferrochelatase activit y i n Ih e bon e marro wi s depressed b

y lead , however , hepati c cell s sho wa compensator

y increas e i n th e activil y o r thi s enzym e (Otrzonsek , 1967) . Thi s ma y explai n som e o r th e clinica l difference s betwee n lea d poisonin g an d acut e porphyria , i n whic h th e principa l effect s o facut e porphyri a ar e i n Ih e liver , whereas thos e o f lea d poisonin g woul d appea rt o be i n ih e bon e marrow . R . Th e Influenc e o r dclta-aminolemlini c Aci d Th e rol e o r th e hem e biosyntheti c precurso rAL A withi n th e bod y ha s bee n Ih e subjec

t o r severa l investigations , owin g t o it s involvemen t i n th e pathogenesi s o r lea d poisoning , acut e porphyri a an d hereditary tyrosin anemi a (Becke ran d Kramer . 1977). Attack s o r acut e porphyri a ar e characterized , amon g othe rsigns , b y ascendin g paralysis , whic h ca n progres s t o quadriplcgi a an d t o respirator y failure. Durin g thes e attacks, th e bloo d an d urinar y level s o r AL A reac h very hig h values . I n lea d intoxica tion , bloo d AL A concentration s ma y doubl e o rmor e (Meredit h e t al. . 1978 ) an d hepati c concentration s o r lea d i n these circumstance s hav e bee n found t o be increase d fourfold (Sukct a et al. , 1975) . I n norma l circumstances , bloo d AL A concentration s ar e i n th e regio n o r 3 >imol/ l (Meredit h e t at. , 1978) . Durin g attack s o l acut e porphyria , th e concentratio n o r AL A i n plasm a ha

s bee n reporte d t o b e as hig h a s 2 4 /ig/m l (20 0 pmol/l) . a t whic h lim e th e AL A concentratio n i n cerebrospina l flui d wa s 2. 8 pg/m l (23 /itnol/l ) (Sweeney c

t al. , 1970) . Thes e concentration s ar e commonl y muc h lowe r(Moor e an d Meredith , 1976 : Perc y an d Shanley , 1977) . I n laborator y animals , hig h level s o fAL A i n Ih e bod y ar c associate d wit h variou s behaviora l changes , includin g som e impairmen t o fvoluntary movement . Thi s effec t has bee n observe d afte rdirec t administratio n o f AL A (Moor e an d Meredith . 1976 : Shanle y e l al. . 1975 ; Yuwile re t al. , 1970) . AL A has als o bee n show n t o have variou s pharmacologica l activitie s (McUillio n e

t al. , 1975 : Loots et al. . 1975 : Becke re t al. . 1976 ) an d indee d t o b e take n u p b

y variou s tissues includin g th e brai n (Becke ret al. , 1974 ; Moor e an d Meredith , 1976 : Shanle y c t al. . 1975) . Som e workers , however , hav e suggested tha t ther e ar e fe wbehaviora l effect s from AL A i n animal s (Shanle y e

t al. , 1975 ) o ri n ma n (Doss . 1976 ) o rloxi c effect s i n mic e (Ken ned y e l al. , 1976 ) o ri n rat s (Picrac h et al. , 1977) . I n othe rexperiments , effect s o r {-aminolevulini c aci d (ALA ) o n socia l behavio rwer e examine d b y elholupica l analysi s t> rencounter s betwee n mal e mice . Thirt y t o forty minute s afte rreceivin g a n injectio n o fALA , mic e explore d th e cag e an d scanne d les s frequentl y tha n saline-injecte d control s an d showe d longe rperiod s o fimmobilit y (Cutle re t al. , 1979b) . Blockag e o r neuromuscula rrunctio n appear s t o be th e predominan t effec t o r AI. A i n those preparation s wher e th e muscl e becomes unresponsive t o nerve stimulation . A separat e toxi c action , possibl y affectin g membran e transport o rmuscl e metabolism , appear s t o predominat e whe n inhibitio n o r muscula r contractio n precedes blockag e o fth e actio n potenlial , at whic h tim e th e muscl e becomes temporaril y unresponsive l o direct electrica l stimulation . Som e o fth e know n action s o r AL A tha t ma y be responsibl e lo rit s effect o n nerve-muscl e functio n includ e a n inhibitio n o fmembran e sodiu m transport (Kale s e l al. . 1971 ) an d a n inhibitio n o r Na'-K ' dependen t ATPas e (Becke re l al. . 1971 ) wil h associate d increas e i n capillar y permeabilit y (McCiillio n e t al. . 1975) . Becke rc t al . (1975 ) found tha t AL A significantl y reduce d th e restin g membran e potentia l o r frog sartoriu s muscle ; thi s i s a n effec t whic h woul d be expecte d fro m a n inhibitio n o fNa* - K * dependen t ATPase . However , Feldma n e t al . (1968) . usin g th e ra t phreni c nerve hemidiaphrag m preparation , obtaine d result s suggestiv e o f hyperpolarization . The y found tha t AL A i n amount s tha t coul d be expecte d i n th e acut e phase o r porphyri a produce d a presynapti c inhibitio n o r th e potassium-augmente d miniatur e endplat e potentia l frequency an d appeare d l o reduc e membran e excitability . I t i s possibl e tha t AL A ma y inhibi t th e transport o r sodiu m i n bot h direction s acros s th e cel l membran e b y blockin g th e channel s throug h whic h sodiu m i s transporte d int o th e cel l durin g propagatio n o r a n impulse , i n additio n t o its effect s o n ATPase . Som e interferenc e wit h io n transport acros s th e cel l membran e wa s indicate d i n th e experiment s o r Cutle rc t al . (1979a ) b y Ih e presence o r polyphasi c actio n potential s tha i occurre d i n frog nerve-muscl e preparation s following applicatio n o r ALA . Inhibitio n o r Na'-K " dependen t ATPas e ma y als o affec t neuromus cula rfonction. Man y differen t experimenta l condition s know n t o inhibi t thi s enzym e deplet e acetylcholin e concentration s o n presynapti c site s (Vizi , i I 

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# tjiSff$$!^frfY 4 < I it " * •• wliv i "i i '.••• •1977) . Suc h a n effec twoul d provid e a n explanatio n forth e ALA-induce d

spontaneou s contraction s o f muscl e an d fo rth e initia l increas e i n muscl e contractio n prio rt o blockage . A furthe rstud y o f

th e mod e o f

actio n o rAL A i s likel y t o provid e som e interestin g informatio n tha tma y she d ligh to n th e rol e o rAI. A i n lea d poisoning . Suc h studie s d o no texclud e th e possibilit y o reffect s from th e metabolite s o rALA . th e monopyrrole s suc h a s porphobilinogen , whic h hav e als o bee n show n t o caus e presynapti cneuromuscula rinhibitio n (Feldma n e t al. . 1971) . Thes e variou s studie s hav e clearl y demonstrate d tha tth e interactio n o r lea d wit h tw o enzyme s o rhem e biosynthesi s result s i n marke d increase s i n bot h urinar y an d hloo d level s o rAI.A . Thi s passe s throug h th e blood-brai n harrie ran d ca n caus e marke d change s i n spontaneou s activit y o rrodents . Th e possibilit y therefor e exist s tha tAL A ca n ac tcithe rdirectl y o rindirectl y o n neuromuscula rfunctio n an d caus e som e o ral l o rth e neurologica l an d behaviora l effect s o f

lea d toxicity .
XII . SUMMAR Y AM ) CONCLUSION S Th e influenc e o f

lea d upo n hem e biosynthesi s an d upo n th e initia l stage s o f hem e degradatio n ha s bee n reviewed . Th e principa l effect s o rlea d ar cupo n th e biosyntheti cenzymes . Th e enzyme s inhibite d ar e {-aminolevulini caci d dehydratase , coproporphyrinoge n oxidas e an d ferrochelatase. Th e greates t diminutio n o f

activit y i s in th e firs to f these , {-aminolevulini caci d dehydratase . A s aconsequenc e o rdepresse d synthesi s o rheme , th e activit y

~ o f th e initia l an d rat e controllin g enzym e o rth e pathway , {-aminolevulini c aci d synthase , i s increased , a s i s th e activit y o rheme-oxygenase , th e first enzym e i n th e biodegradativ e sequence . A s aconsequenc e o rthes e change s i n enzym e activit y ther e i s a n increas e i n th e productio n an d excretio n o rth e intermediate s o rth e biosyntheti cpathway , {-aminolevulini cacid , coproporphyri n an d protoporphyrin . Th e change s i n thes e intermediate s hav e bee n use d togethe r wit h {-aminolevulini caci d dehydratas e a s secondar y indice s o f lea d exposure.Sinc e synthesi s o f hem e i s depressed , s o als o i s th e synthesi s of > hemoprotein s suc h a s cytochrom e P-45 0 an d hemoglobin . Depressio n o f cytochrom e P-45 0 b y lea d wil l caus e alteration s i n hepati cdru g metabolis m i n bot h animal s an d man . Anemi awil l als o develo p durin g lea d poisonin g du e t o thre e factors. Thes e ar e depresse d hem e synthesis , altere d globi n synthesi s an d re d cel l fragility resultin g i n hemolysis . Finally , becaus e lea d poisonin g i s s o simila ri n presentatio n t o th e acut e porphyrias , thes e ar e compare d an d contrasted . I ti s considere d possi bl e tha tth e associate d neurologica l manifestation s o rthes e disease s occu ri n par tthroug h th e overproductio n o rth e porphyri n precurso r{-aminolevul ini cacid , whic h i s show n t o b e neuroactive .
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