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•• it^-W3w*rar»»aw»7i»'*>«m^^J . JHLIT.-'IJ T*»-''"' ' —•» • '• I III 

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Interact. 12:19. 

Lead and Heme 

Biosynthesis 

Michael R. Moore. Peter A. Meredith aad Abraham Goldberg 

TABLE OF CONTENTS 

I. INTRODUCTION - 8 0 

II. THE HEME BIOSYNTHETIC PATHWAY 80 

A, Dqlia-aminolevulinic Acid Synthase 83 

B. Oelia-AminolovulMiic Acid Dehydratase 83 

C Uroporphyrinogen 1 Synthase and 

UroporphyrinogenAitynthetasa 83 

O. Uroporphyrinogen Doctvhoxyl-ise 84 

E CnprotKwphyrinoaen OxrdaM 84 

F Protopofphyrinogen Oxidase 84 

G. Fnrrochelatase and Heme Oxygennse 84 

lit. CONTROL OF THE HEME BIOSYNTHETIC 

PATHWAY .' 85 

IV. SOURCES OF LEAD EXPOSURE 88 

V. LOCALIZATION OF LEAD WITHIN THE BODY 

VI. THE EFFECTS OF LEAD ON THE ENZYMES OF 

THE HEME BIOSYN THETIC PATHWAY 89 

A. ALA Dehydratase 89 

B. Coproporphyrinogan Oxidase and Ferrochelatase 2 .._. 

C. ALA Synthase and Heme Otygenae* 91 

D. Other Effect* 92 

Vlt. CHANGES IN THE PRODUCTION AND EXCRE-

. TION OF PATHWAY INTERMEDIATES DURING 

LEAO EXPOSURE 93 

A. Delta- aminotovulintntt Acid 93 

B. Porphobilinogen 9S 

C. Coproporphyrin and Protoporphyrin 95 

VIII. THE DIAGNOSTIC USE OF EXCRETION AND 

ACTIVITY PARAMETERS 96 

A. Delta-aminolevulinic Acid Concentration* in 

Urine 97 

B. Blood Concentrations of ALA 98 

C Urinary Coproporphyrin 99 

D. Blood Protoporphyrin 100 

E. Eiyihrocyte delta- aminolevulinic Acid 

Dehydratase 100 

F. Inter comparison of Ihe Relative Value of 

Heme Bioiynthatrc P.iramelvra in Assesi' 

ment of Lead Exposure 102 

G. Hemoglobin 103 

IX. CHANGES IN DRUG METABOLISM DURING 

LEAD EXPOSURE 103 

A. Animal Studies 104 

B. Human Studies 108 

7« 
, , W JlSS, »*.'**, VTT* •• 

X. THE ANEMIA OF LEAD POISONING 107 

XI. A COMPARISON OF LEAD POISONING AND 

ACUTE HEPATIC PORPHYRIA 109 

A. General Considerations 109 

B. The Irrfluenca of Oetta-aminotvtlltnie Aeld 112 

XII. SUMMARY AND CONCLUSIONS 114 

I. INTRODUCTION 

The effect of lead on heme biosynthesis has been long recognized. Indeed, a 

great similarity has been noted between some aspects of lead poisoning and 

the symptomatology of acute porphyrias. Hematoporphyrinuria was noted 

hy Stokvis (1895). Pcrulz (1910) and Gnetzl (1911) in lead-poisoned rabbits, 

l.acnnec (1831) noted thai patients who died oflead poisoning showoil thinness of blood and pallor uf tissues on postmortem examination. Binnchdijk 

(cf. Stokvis. IS95) first noted the occurrence of excess porphyrin in the 

urine of a patient with lead intoxication and in 1927 l.cibig suggested thai 

the rise in porphyrin excretion during lead poisoning was due lo the action 

of lead upon hemoglobin synthesis in Ihe hone marrow, (iroctpass (1932) 

showed that enhanced urinary porphyrins were due principally to the elevation in coproporphyrin series III isomer. More recently. Ilaeger (1957) 

found excessive urinary excretion of {-aminolevulinic acid during lead 

intoxication, ll is cl<:ar from these early pointers that lead has very drmnatic 

effects upon the biosynthesis of heme. 

II. THE HEME BIOSYNTHETIC PATHWAY 

The origins of heme biosynthesis lie in the citric acid cycle and in normal C, 

amino acid metabolism. The elucidation of the early sleps of the heme 

biosynthetic pathway was the result of an elegant set of isotopic tracer 

studies carried out by Shcmin (1955). In the initial slage of the pathway, 

succinate as succinyl CoA combines with glycine, under the aegis of the 

initial enzyme or the pathway, {-aminolevulinic acid synthase (EC 2,3.1.37) 

and the cofactor pyridoxal phosphate to form the first ofthe intermediates 

of the pathway, {-aminolevulinic acid. In the next stage of the biosynthetic 

pathway two moles of {-aminolevulinic acid condense lo form the monopyrrole. porphobilinogen, by the action of the enzyme {-aminolevulinic acid 

dehydratase (porphobilinogen synthase) (RC 4,2.1.24). In the next stage 

of biosynthesis, four moles of porphobilinogen condense, cyclize and isomcrizc to form the first of the porphyrins, uroporphyrinogen, as the three 

isomer. This is carried out by the concerted action of two enzymes, 

uroporphyrinogen I synthase (porphobilinogen deaminase) (EC 4.3.1.8). 

and uroporphyrinogen cosynthase. In circumstances where there is a defi-

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Heure I. The heme hiossnthctic pathway. Heme hiossnlhesis within Ihr cell starts in the 

mitochondrion wilh ihe initial anil rale limiting enzyme AL A synthase. Synthesis Ihen passes 

into the cytoplasm and rclurns once again Hi the mitochondrion Tor His' final three stapes in Ihe 

pathway."Control of the ptlhway is hy negative feedback hy heme upon AL A synthase. The 

enrsmes of the pathway are shown within Ihe open squares and the intermediates of Ihe 

p.ithwav follnw the heavy arrows to Ihe final product, heme. Each of ihe intermediates: may he 

used in the siihw|ticnt production nf the following component of Ihe paihsvay. escepling the 

series I porphyrinopens. uroporphyrinogen I and coproporphyrinogen I. which may not he 

utilized beyond the stage of coproporphyrinogen I and are therefore obligatorily excreted. 

Abbreviations: ALA . iaminolcvulinic acid: PBG. porphobilinogen: COOII . cathoxyl. 

ciency of uroporphyrinogen cosynthase. series I isomer is formed. By successive decarboxylations of the octacarboxyllic porphyrin ringjlhrough Ihe 

hepta. hexa and penta-carboxylic porphyrins, the^tetracarboxylic 

coproporphyrin is formed by the reaction catalyzed -by^ the enzyme 

uroporphyrinogen decarboxylase (EC 4.1.1.37). At this stage the reaction 

re-enters the mitochondrion. whereMCoproporphyrinogen' 'III is decarboxylated by coproporphyrinogen oxidase (EC 1.3.3.3) to'/orm protoporphyrinogen IX. which is a dicarboxylic divinyl porphyrin -Itftjie reducing milieu of the mitochondrion. pfotoporphyVinogen IX"*is oxidized to 

protoporphyrin by protopb'rphyrnogen oxidase. The final step or the 

pathway is the insertion Vf-fcrrous iron into protoporphyrin by ferrochelatase (EC 4.99.1.1) to form heme (Fig. I). The one other component of this 

sequence that should be mentioned is the first or the biodcgradalive steps of 

heme. In this, by splitting of the heme ring with loss or iron, heme 

oxygenase (EC 1.14.99.3) produces bilirubin (Table I). 

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Table I. The enzymes of heme biosynthesis and biodcgradati.w 

Common namc(s) 

i-Amtnolevulinatc synthase 

Porphobilinogen synthase 

4-Aminolcsulinatc dehydratase) 

Uroporphyrinogen I synthase) 

Porphobilinogen deaminase) 

Uroporphyrinogen i cosynlhctase 

Uroporphyrinogen decarboxylase 

Coproporphyrinogen oxidase) 

Coproporphyrinogcnase) 

Protoporphyrinogen oxidase 

Ferrochelatase) 

Heme synthetase) 

Heme-oxygenase 

Abbreviation Systematic name 

ALA.S , Succinyl-CoA: Glycine C-Succinyltransfcrase 

ALA. D 5 Aminoevulinatc hydro-lyasc 

URO.S Porphobilinogen ammonia-lyase "r"' "' 

URO.CoS • Unclassified 

URO. D ' Uroporphyrinogen 3 carboxy-lyase 

COPRO.O Coproporphyrinogen: Oxygen oxidoreductase 

PROTO.O Unclassified 

FERRO.C Protohcme fcrro-lyase 

HEME.O . Heme: NADPH : Oxygen oxido-reductasc 

Enzyme 

classification 

nomenclature 

2.3.1.37 

4.2.1.24 

4.3.1.8 

4.1.1.37. 

1.3.3.3 

4.99.1.1. 

1.14.99.3 

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porphobilinogen molecules. The action of both URO synthase and 

uroporphyrinogen Ml cosynthase catalyses the hcad-to-hcad condensation of 

the porphobilinogen molecules. This results in the formation of an altered 

enzyme-bound dipyrrylmethane which is specifically incorporated into 

uroporphyrinogen II I (Frydman et al., 1976; Battersby and McDonald, 

1976). 

D. Uroporphyrinogen Decarboxylase 

The final cytoplasmic step of heme biosynthesis involves tin: decarboxylation of the octa-carboxylic uroporphyrinogen to the tetia carbo.xylic 

coproporphyrinogen III . The decarboxylation catalyzed by uroporphyrinogen (URO) decarboxylase is a stepwise process, the acetic acid 

groups being dccarboxylatcd in a sequential clockwise fashion starting 

u ilh that on ring II and followed by those of Ihe A, B ant) (J rings (J.-irkson 

ct al., 1976). The specificity of URO decarboxylase is for Ihe scri-.--; III 

isomer of uroporphyrinogen which is decarboxylatcd twice as rapidly as the 

scries I isomer, but it will decarboxylate the scries I isomer to pmducc 

iroproporphyrinogcn I which is excreted as coproporphyrin I (Fig. 2). 

K. Coproporphyrinogen Oxidase 

At this stage in the pathway the reaction scries rc-cntcrs the mitochondrion 

for the conversion of coproporphyrinogen II I to protoporphyrinogen IX by 

the decarboxylation of two propionic side chains lo vinyl groups. The 

enzyme coproporphyrinogen oxidase catalyzes the conversion of coproporphyrinogen II I to protoporphyrinogen IX. The mechanism for this 

reaction is not altogether clear, but the evidence is compatible with three 

closely related mechanistic alternatives (Akhtar et al., 1976). whereby there 

is a stepwise decarboxylation of the propionyl groups to vinyl groups with a 

monovinyl porphyrinogen existing as an intermediate. 

F. Protoporphyrinogen Oxidase 

Until recently, indirect evidence suggested that the oxidative conversion of 

protoporphyrinogen IX to protoporphyrin IX in the reducing milieu of the 

mitochondrion was controlled enzymatically (Porra and Falk. 1964). 

Jackson ct al. (1974) conclusively demonstrated that this was true. The 

enzyme protoporphyrinogen oxidase has subsequently been isolated from 

both microorganisms (Poulson and Polglase, I97S) and mammalian tissue 

(Poulson, 1976). 

G. Ferrochelatase and Heme Oxygenase 

The final step in this biosynthetic pathway is the insertion of ferrous iron 

into protoporphyrin IX by ferrochelatase to form Heme. This enzyme is 

widely distributed in nature and as a particulate enzyme is found associated 

PORPHYRIN "OUPHYIINOCt N 

ACE « 0 [ 

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3 ' ' l-eopinnyl Acplyl 

Cipropo>pl,yf infngrn)! M*il,yl Mtihy l " M^>hyl Pmpionyl 

J Propipnyt MetKyl 

PiO'opo,pl,ytSn S* " Vinyl " 

tigure 2. The structure of the porphyrins ami porphyrinogens. By Attachment of the lipands 

sll'nvn ill the positions A to II roiiml the porphjrin or porphyrinogen n'tclcus. each or the 

components of the liiosynlhetic pathway of heme is shown. Heme itself is formed hy insertion 

of ferrous iron into the center of the protoporphyrin s) nucleus. The porphyrin is the osidired 

form ofthe porphyrinogen, which is the normal biosynthetic intermediate. 

with the inner mitochondrial membrane in animals (Mackay et al.. 1969) 

and bound to chromalophorcs in microorganisms (Neuberger and Tait, 

1964). The mechanism of this enzymic reaction has not yet been clearly elucidated, bul of all the factors affecting ferrochelatase activity lipids, and 

especially phospholipids, seem to have the greatest effect (Yoneyama et al., 

1969). Heme oxygenase catalyzes the initial rate-limiting step of heme degradation by preferentially splitting the tetrapyrrolc between the A and D 

rings, thus producing the linear tetrapyrrolc bilirubin 9n with consequent 

loss of iron and of carbon monoxide. This reaction which is microsomal 

requires oxygen and NADPII . 

III. CONTROL OK THE IIKME RIOSYNTIIKTIC PATHWAY 

In normal circumstances, this is a highly efficient and coordinated pathway 

producing only sufficient amounts of the intermediates and ultimately of 

heme to service requirements for hemoglobin and olher hemoprotein syni i:, 

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VI. THF. EFFECTS OF LEAD 

ON THE EN7.VMES OK THE HEME BIOSYNTHETIC PATHWAY 

• fltslCOrpuKHI 

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figure 4. Distnhution of lead ssithin the body. The three sources of lead intake are shown 

here. Dietary intake will result in about 10'J adsorption from the gut. Pulmonarv intake will 

result in about 40-, absorption from the lungs. Percutaneous ahsnrption will result pencr illy in 

a negligible intake through the skin, excepting where lead is in the form of an alksl lead compound which may he absorbed up to 1% or applied material, following absorption. Lad it 

transported by Ihe blood lo Ihe soft tissues and to the bone. Greater, than 90^ of the total body 

lead burden will he found in bone, wilh relatively high concentrations in liver and kidncss Urinary excretion accounts for Ihe greater source of lead loss, although some may also he e'screted 

in the feces. Red blood corpuscles shown by an open circle: Lead shown hy a closed triangle 

Lead has many diverse biochemical efTccts, all of which are of a deleterious 

nature. No evidence has been presented for an essential function for lead in 

metabolism. Lead has a valence shell electronic structure of 6s* 6p' and normally exhibits a valence of two. The vacant 6p orbitals may be filled by the 

formation of covalcnt bonds; this occurs most readily in biological systems 

with sulphur atoms in proteins. These biochemical effects, therefore, all 

probably relate ultimately to the capacity of lead to combine with specific 

biochemical ligamls. These include sulfhydryl groups, amino groups, 

carboxyl groups, pheno.xy groups and imidazole residues. In such circumstances, lead might be expected to alter tertiary structures of biochemical 

molecules and, therefore, alter or destroy their biochemical function. Consequently, lead can change enzyme activity and destroy the structure-function relationships of nucleic acids. 

It is clcnr from many studies Ihal lead can markedly inhibit mitochondrial respiration. In addition lo lite defective structure of their membranes, 

isolated mitochondria show impaired oxidative phosphorylation and. ultimately, impaired mitochondrial respiration. 

Of the complex of enzymes that synthesise succinyl CoA from n-keto 

glutamic the dilhiol enzyme lipoamide dehydrogenase is markedly 

inhibited by lead. This inhibition is probably representative ofthe effects of 

lead on many enzymes either containing sulfhydryl groups or activated by 

sulfhydryl groups. 

A. ALA Dehydratase ____ _ _ 

It is clear from consideration of excretion patterns during lead exposure 

that specific stages during the biosynthetic pathway are particularly vulnerable to the effects oflead. Studies have now been carried out on most enzymes 

or the pathway and have indicated both in animal and in human studies just 

which stages are most susceptible to change. The most discussed enzyme in 

this respect is {-aminolevulinic acid dehydratase which, during lead 

exposure, has clearly been shown to be markedly inhibited (Hernberg and 

Nikkancn. 1972) (Fig. 5). This results in excessive production and excretion 

of 6-aminolevulinic acid. The mechanism of this inhibition remains unclear, 

but it is associated with alterations in the concentration of free sulfhydryl 

groups in biological systems. The availability of concentrations of other 

metals such as zinc may also contribute, since zinc has been shown to 

activate AI.A dehydratase (Meredith and Moore. 1978). 

Organic lead poisoning is rare: thus there arc only a small number of 

studies showing its effects on the heme biosynthetic enzymes. What studies 

there are suggest that the effects of organic lead on these enzymes are 

similar lo those of inorganic lead. Millar et al. (1972) showed that diethyl 
-xir-,^*^rA*v-*rsrwv^-s'^-<:^,r*wr**^ 

thesis within the cell. In some circumstances, control of the pathway can be 

so altered by gross disturbances of the biosynthetic sequence as to produce 

very large quantities of redundant precursors which must then be excreted. 

L-.:-'l poisoning is one circumstance where such an effect can take place. 

Several features combine to make the heme biosynthetic pathway 

easily, and accurately controlled. The first important feature is that heme 

acts by feedback repression and inhibition upon the initial enzyme of the 

pathway, ^-aminolevulinic acid synthase. This step then becomes the ratecntrolling step of Ihe pathway. This control is aided by the short half-life 

ot this enzyme: it is about I lo I 'h hr in mammalian systems (Moore and 

Goldberg. 1974). Control is further aided by membrane permeability 

considerations, since during the sequence of the pathway (he product passes 

from the mitochondrion to the cytoplasm and then back into the 

mitochondrion again. If at any stage in the pathway oxidation docs take 

place of the porphyrinogen lo the porphyrin, such porphyrin must be excreted since it cannot be further used in the biosynthetic sequence, l-qually, 

any deficiency of the enzyme uroporphyrinogen cosynthase leading to 

production of scries I isomer porphyrin will require that such porphyrin is 

also excreted since it may not be used beyond the slage of coproporphyrin I 

in normal biosynthetic mechanisms. It is clear, therefore, that any circumstance that can alter Ihe relative proportions of activity of these enzymes 

will alter the quantity of precursor produced during the progress of the 

pathway. 

IV. SOi;R{ F.S OK LEAD EXPOSURE 

There are multiple sources or lead exposure within the environment. Most 

of these are summarized in Figure 3 and all derive ultimately from the lead 

in the earth's crust. For modern man. the greatest exposure takes place 

through the food that he eats and the water that he drinks, although in 

specific circumstances inhalation of air which is contaminated with lead or 

uptake of dust from industrial processes may represent important exposure 

vectors (Fig. 3). 

V. LOCALIZATION OF LEAD WITHIN THE BODY 

The actual sites within the body in which lead is deposited are as important 

a feature as. and part of its effect upon, the heme biosynthetic pathway. 

Although during the absorptive processes lead is found in excess in Ihe 

blood, following absorption it is rapidly transferred to. soft tissues and ultimately ends as depot lead in bone. Within the bone lead is relatively inert, 

yet in excess of 95% of the whole body lead burden will be found in the 

bone. In these circumstances, lead is readily available to the marrow. In 

Figure J. The vectors of human lead exposure. The sources from which man may be exposed 

to lead are shown hy the arrows, w-ith the heavier arrows indicating the greater degress of 

exposure. The principal source of human uptake of lead is through food and water consumption. Absorption may also take place through the air breathed, by percutaneous absorption of 

alkyl lead compounds in gasolines and through various sources of contamination. 

consequence the processes of red cell growth and maturation and. of course, 

formation of hemoglobin for these cells will be inhibited. In the soft tissues, 

lead is found in greatest concentrations in the kidney, the principal lead 

excretory organ. Lower but significant concentrations of lead are also found 

in liver and in spleen. The distribution of lead within the body is shown in 

Figure 4. It may be noted that although only 10% or ingested lead is 

absorbed, up to 40% of inhaled lead will be taken in by the pulmonary 

route, providing the particle size is sufficiently small. Percutaneous absorption of lead is unlikely to play any important role in its uptake unless the 

lead exists in a lipid soluble form, such as the alkyl lead compounds 

(Fig. 4). 

•I Ifc:

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

«H 

ERYTHROCYTE 

ALA.D 

[nmol ALA/mtn/ 

ml ,.b..| 

2 3 

BLOOD Pb(wmol/l| 

Figure 5. The relationship between blood lead concentrations and erythrocyte &-aminnlevu-

~linic~acid dehydratase'activity. As blood lead concentrations rise th h'umans. so the activity~bf 

erythrocyte A LA dehydratase falls exponentially, with the greatest rate of decrease being seen 

al lower blood lead concentrations. For this graph, the mathematical relationship is erythrocyte ALA.D - 58le- ,

""~ " •—' + 0.06. 

lead depresses ALA.D activity while Beattie et al. (1972). in investigations 

on four subjects exposed to lead alkyls. demonstrated a marked depression 

in erythrocyte ALA.D activity and a modest elevation or erythrocyte 

protoporphyrin (PROTO). but no elevations in urinary ALA and COPRO. 

B. Coproporphyrinogen Oxidise and Ferrochelatase 

The next stage in the pathway that is clearly inhibited by lead is at the level 

of coproporphyrinogen oxidation as shown by excessive urinary excretion of 

coproporphyrin. Recent studies have shown that lead exposure in man 

and animals results in highly significant depression of activity of 

coproporphyrinogen oxidase within the mitochondrion (Campbell et al., 

1977). The last enzyme ofthe pathway, ferrochelatase. has also been shown 

lo be inhibited by lead and may contribute to the sideroblastic types of 

anemia that develop during lead poisoning (Campbell et al.. 1977) and, in 

addition, to the marked rise in protoporphyrin synthesis (Fig. 6). 

C. ALA Synthase and Heme Oxygenase 

The activity of two enzymes in the biodegradative and biosynthetic sequence 

are known to be elevated during lead exposure. Recent animal experiments 

have demonstrated that the activity of heme oxygenase is raised by lead 

presentment (Maines anil Kappas, 1976a: Goldberg ct al.. 1978). The net 

effect of diminished rcrrnchelalasc activity and raised heme oxygenase 

activity is to lower the availability or free heme concentrations within hiolugical systems by virtue of the feedback control of heme upon the activity 

of the initial and ratc-limiling enzyme of the pathway. The result is that the 

activity of this enzyme o-nminolevulinic synthase is raised during lead 

exposure (Maxwell and Meyer. 1976). presumably in an attempt to compensate for diminished heme production in these circumstances. The combination of increased activity of ALA synthase and decreased activity of 

AI.A dehydratase can account in kinetic terms lor the very marked increase 

in circulating and excreted AI.A during lend exposure (Meredith el al.. 

1978). These changes in heme biosynthesis are detailed in Fig. 7. 

Figure 6. The effects of lead exposure on coproporphyrinogen oxidase nnd ferrochelatase. 

The activity of these two cn/ymes shows a pronounced diminution of activity after industrial 

lead exposure. Coproporphyrinogen oxidase. COPRO.O; units, n mol protoporphyrin 

utili/ed/g nrotein/hr; ferrochelatase, FKRRO.C: units, percentage, uptake of "Fe into 

heme/hr. 

'.'-ii?- j'.n2J..*.t;- ;••-! 
•ffttct of lead on 

•ntyn* activity 

ALU « -

(urlM) II 

COPRO * -

lurlnel 

PR0T0 « -

(blood) 

GLTciire • socciim . Co* 

I MA SynthaM 

1-MUKOLAEVtrLMIC ACID (MA) 

AIA Dehydratase 

PORPHOBILIMOCt* (PBO) 

URO 1 SynthaM 

0R0 Coaynthaae 

UROPORPHYRINOGEN:!i! (ufo) 

r 

I URO Oecarboxyla«« 

COPROPORPHYRINOGEN'III (COPRO) 

I COPRO Oxidate • 

PR0T0P0RPHTRJNOGCN 

Protoporphyrlnogan 

Oxidate 

PROTOPORPHYRIN IX (FROTO) 

ra-rrochalatata 

~l •• 

HAEM 

Figure 7. The efTccts of lead on heme biosynthesis. Lead inhibits three enzymes of heme 

biosynthesis: these are ^-aminolevulinic acid dehydratase, coproporphyrinogen oxidase and ferrochelatase. As a consequence of decreased heme production, there is a rise in the activity of 

the initial ratc-limiting enzyme ^-aminolevulinic acid synthase. As a function of these blocks in 

heme biosynthesis and the increased input of ALA into Ihe biosynthetic pathway, three intermediates of the pathway are produced in excess. The first ti o-aminnlcvulinic acid, which is 

excreted in the urine: the second is coproporphyrin. also excreted in the urine Anally, 

protoporphyrin is produced and found in excess in the erythrocytes. 

D. Other Effects 

Although these are the principal points at which lead may affect the 

biosynthetic pathway, other features have been noted during lead exposure. 

Uncommonly, porphobilinogen concentrations may'rise: this has been 

ascribed to alterations in the activity of uroporphyrinogen I synthase 

(porphobilinogen deaminase). Changes in activity of uroporphyrinogen I 

synthase have been suggested as a secondary control point within the 

biosynthetic pathway (Brodie el al.. 1977). In circumstances where the 

activity of this enzyme is raised, one would not expect elevated concentrations of porphyrin precursors. In lead exposure, however, lowered activity of 

AI.A dehydratase is coupled with an increase in uroporphyrinogen I 

synthase, which will allow raised concentrations of {-aminolevulinic acid but 

will generally not show elevated concentrations of porphobilinogen (Brodie 

et al., 1977). Intriguingly, recent studies have shown that the activity of this 

uroporphyrinogen I synthase inhibited during in vitro lead exposure may be 

protected from the effects of load by availability of pteridine derivatives 

such as pteroyl hexaglutamate (Piper and Tephly. 1974: Piper and Van 

l.icr, 1177). Other studies have shown that at higher concentrations of 

lead the decarboxylation of uroporphyrinogen lo coproporphyrinogen 

is also inhibited (Krcimer-Birnhaum and Orinslcin, 1965). One enzyme 

which is almost certainly not affected by increased lead exposure is 

uroporphyrinogen cosynthase. as evidenced by the excretion of scries III 

isomer porphyrin during lead exposure, rather than excretion or series 1 

isomer porphyrins (Fig. 2). 

VII. CHANGES IN Till-. PRODUCTION AND EXCRETION OF 

PATHWAY INTERMEDIATES DURING LEAD EXPOSURE 

A. Della-aininolciulinie Acid 

Studies in humans of the kinetic relationships between lead exposure, 

the activities or 6-aminolevulinic acid synthase, .-aminolevulinic acid 

dehydratase and blood ALA concentrations have shown a positive 

exponential relationship of ALA.S and blood lead (Fig. 8) and a negative 

exponential relationship of ALA.D and blood lead (Fig. 5). The association 

between blood ALA and ALA.D and their association consequently with 

blood lead is in the form of a power curve. Similarly, there is a power relationship between blood AI.A and excretion of urinary ALA (Meredith et 

al., 1978). 

These results have demonstrated that in lead intoxication there are 

large increases not only in urinary concentrations of ALA but also in circulating levels of ALA. This elevation is brought about by two factors: these 

are, firstly, the depression of ALA.D and. secondly, an elevation ofthe ratelimiting enzyme of heme biosynthesis ALA.S. Depression of ALA.D by 

lead is well documented, but elevation of ALA.S has only recently been 

shown in lead intoxication in man (Takaku et al.. 1973; Campbell ct al., 

1977). 

The'relationship of raised urinary ALA and erythrocyte ALA.D shows 

that the consideration of such a relationship gives limited metabolic 

information. The absence of a straight-line relationship between blood ALA 
*.*~.*r-~-.j~.*** •*,-.-•..-r«"-;,

,—^-.»*.,<,**i«l'"'r. 

•Mv*,r*MF*s«.--

94 Least I o.xicity 

"0 I J I 4 J « 

Blood Lr>.-id ',)imdl/\\ 

Figure H. The relationship between blood lead concentrations and leucocyte i-aminoloulinic 

acid synthase activity. As blood lead concentrations rise, so Ihe activily of ^-aminolevulinic 

-acid-synlhase-riscs exponentially.-In-lhis graph.-normat-subjects are-shou-n as open.circles and_ 

lead exposed workers by closed squares. The mathematical form of this equation is 

Leucocyte ALA.S . 4» Je •""-"" • + W.J. 

and urinary ALA suggests that, at higher blood concentrations of ALA, a 

greater proportion is excreted into the urine. This results in a plateau, of 

blood ALA concentration when blood lead concentration is in excess of 

3 Mmol/1. At this concentration, urinary ALA shows a rapid rise (Figs. I0A 

and IQB). Reduced renal tubular reabsorption has been considered to be a 

contributing factor in this rise (Druyan et al.. 1965). However. Cramer and 

coworkers (1974) showed that ALA clearance is similar to the glomerular 

filtration rate, although other amino acids were excreted in excess during 

lead intoxication. There is then an accelerating rise in ALA excretion 

consistent with reduced tubular reabsorption. Thus, below blood ALA 

concentrations of 4 fimol/l. the relationships of ALA in urine and blood are 

linear, ll is only when blood ALA concentrations rise above this value that 

there is a rapid rise in urinary ALA excretion, possibly associated with 

decreased tubular reabsorption. Below blood lead concentrations of 2 

pmol/l. the relationships of ALA in urine and blood are linear. It is only 

Lead and Heme Biosynthesis y:> 

when blood lead concentrations rise above this value that inhibition of ALA 

dehydratase and, consequently, heme biosynthesis is sufficiently great to 

depress synthesised free heme, induce ALA synthase activity and cause 

these accelerating rises in synthesized ALA. 

The interrelationships of blood lead, leucocyte ALA.S. erythrocyte 

ALA.D and blood ALA are of considerable metabolic importance, provided 

one assumes that the concentrations of these parameters parallel those in 

other tissues. This is a reasonable assumption if the individual is in a steady 

state condition. These relationship* suggest that there are "critical" tissue 

lead concentrations, represented by a blood lead concentration of 

approximately 2 /imol/l and an erythrocyte ALA.D activity of approximately 18 nmole ALA utilized per minute/ml red blood cells. Above this 

concentration and In-low this activily. the depression in heme synthesis is 

sufficient to raise the activity of ALA.S. the rate-limiting enzyme of heme 

biosynthesis, by negative feedback (Fig. 9). 

II. Porphobilinogen 

It is not certain in what manner lead poisoning may influence the urinary 

excretion of PBU. Most studies suggest that it will be normal, although is 

some cases it has been reported as raised (Waldron and Stofen, 1974). The 

current concept of control of heme biosynthesis would suggest that the 

concentration should be normal in urine and plasma to correspond with 

raised activity of uroporphyrinogen I synthase (Brodie et al., 1977), 

although the activity of this enzyme has been reported to be inhibited by 

lead (Piper and Tephly; 1974). -•- - . 

C. Coproporphyrin and Protoporphyrin 

Excessive urinary porphyrin excretion has long been recognized. This is 

caused principally by the rise in coproporphyrin. mainly as the series II I 

isomer. Subsequently, it has been shown that other porphyrins may be 

detected in increased amounts in lead intoxication. Increased concentrations 

or protoporphyrin may be found. So also can the concentration of 

uroporphyrin rise in severe cases of lead poisoning, with associated 

increases in intermediate porphyrins of the biosynthetic pathway (Waldron 

and Stofen. 1974). 

A notable feature of lead poisoning is the elevation of erythrocyte 

porphyrin levels. Most of this is in Ihe Torm of protoporphyrin, although 

erythrocyte coproporphyrin is also raised. The elevation or erythrocyte 

protoporphyrin has received considerable attention as a bioanalytical index 

of lead exposure. This has resulted in the publication of recommendations 

for the use of protoporphyrin as a primary screening test in lead intoxication (Center for Disease Control, 1975). Protoporphyrin is found both in the 

free stale and as a zinc chelate. Both of these fluoresce under ultraviolet 

'I.! 

rftsSr"-

i

':W't<Srs'--if.....-1v, 

'•''IE K&4wfer^- i 
rfaswsm-vs.ir/ss*-

96 Lead Toxicity 

MOOf 

WOO 

LEUCOCYTE 

ALA.S 

^•nol ALA/K/ B protein 

800) 

to 20 30 40 

ERYTHROCYTE ALA.D 

•nmol ALA /mi n / ml tbcj 

50 

Figure 9. The relationship between erythrocyte 4-aminotevulinic acid dehydratase activity and 

leucocyte ^-aminolevulinic acid synthase activily. Corresponding to Ihe inhibition of ALA.D by 

lead and the induction of ALA synthase by lead. Ihe interrelationship of these two enzymes is 

in ihe form of a power curve. In this graph, normal subjects are shown as open squares apd 

lead-exposed subjects as closed squares. The mathematical form of this equation is 

c J t .. . , . „ .. . fleucocvte ALA.S - 99.2 Erythrocyte ALA.sD-SSlf P - " ' ' c 

48 3 f 

light and result in the development of fluorocytes. that is. erythrocytes 

which show red fluorescence under ultraviolet light (Whitaker and Vietti. 

1958). 

VIII. THE DIAGNOSTIC USE OF 

EXCRETION AND ACTIVITY PARAMETERS 

It is clear from the various features of the effect of lead upon Ihe 

biosynthetic pathway that the changes in both the activity of enzymes and 

the production and excretion of intermediates of the pathway can be used as 

secondary biological indicators or lead intoxication. Although the earliest 

references to the biological effects of lead upon heme biosynthesis referred 

to increases of porphyrin in the urine, it is now certain from consideration 

of available evidence that such changes are certainly not the most sensitive 

or the most easily measured indices of exposure, lo r the purposes of this 

section, each of the different substances and enzymes and their uses will be 

reviewed in turn. These are the concentrations of o-aminolevulinic acid in 

blood and in urine, (he concentrations of coproporphyrin in urine, the 

concentrations of protoporphyrin in the blood, the activity of o-aminolevulinic acid dehydratase in blood and. finally, comparison of Ihe relative 

efficiency of each of these. Also included are the effects of lead on the 

concentration of hemoglobin in the blood, although it is certain that a very 

severe degree of lead intoxication must occur for changes in the concentration of hemoglobin to he found. 

A. Deltn-aminolewdinic Acid Concentrations in Urine 

The early studies of llae<?.ei-Aronsen (I960) demonstrated clearly that 

measurement of ^-aminolevulinic acid in the urine was a useful diagnostic 

test for lead exposure in lead workers. The method,by which ALA is 

measured in these circumslauccs has always been the colorimetric technique, whereby rS-aminolevulic acid is condensed with another compound, 

usually acclyl acetone, lo form an l-hrlich's positive pyrrole, which may 

then form a pink color quantitatively wilh paradimethyl aminohcnzaldchyde 

(l-hrlich's reagent). When the concentrations of urinary ALA and blood 

lead are compared, a linear relationship which intercepts the blood lead axis 

at values above the origin is seen. In the initial stages of the graph, changes 

in concentration are so small as the be of little use. However, when blood 

Table 2. The concentrations of lead, porphyrins and porphyrin precursors in 

normal and lead-poisoned humans 

Normal 

subjects 

Lead-Poisoned 

subjects 

Blood lead fimo\/\ 

, (Mg/100 ml) 

j Vrinary AI.A minol/mol creatinine 

(nmol/l) 

| Blood ALA pmol/l 

] Urinary coprrtporphyrin 

' umul/rmil creatinine 

:

 (nmol/l) 

Erythrocyte protoporphyrin 

(nmol/l) 

0-1.75 

(0-40) 

0-2.5 

0-43 

0.-3.5 

0-12.5 

0-220 

0-0.75 

>4.0 

(>80) 

>4.0 

>M> 

>5.0 

>40 

>300 

> I 5 

u 4$ct#y&.'lV' 

Jt<:zK* &;^A:.^.'-'vv * 

I tat '* '••'"',V,-:' ''"•'-

L-Jir^'s^'V- • 

''».-?.:•. wl?sV ^ 

fe 
98 Lead Toxicity 

lead concentrations exceed normal limits. I.e., greater than 40 <ig% (2 /iM), 

urinary ALA concentrations show some rise and continue to do so as blood 

concentrations of lead rise (Fig. 10). Normal values for ALA in urine are 

shown in Table 2, together with the range of values in a series of industrially 

exposed workers. 

B. Rlood Concentrations of AI.A 

The concentration of/S-aminolevulinic acid in the blood may also be used as 

an index of lead exposure, although the concentrations likely lo be achieved 

are certainly not as great as those in acute porphyria in attack. In the 

normal person, blood concentrations of ri-nminolcvulinic acid arc small, but 

following industrial lead exposure they rise rapidly. The mclhoi!- for 

measuring blood AI.A are similar to those for urine, except that the blood 

initially must be treated with iodo-scctamidc to complex interfering 

sullhydryl groups, after which ALA'wiay be extractcil into acid and 

3 

< 

* Blood Lead [uMi 

Figure 10. the relationship between blood lead and hlood and urinary ^-aminolevulinic acid. 

As blood lead concentrations rise, so the concentration of the urinary excretion of 6-aminolevulinic acid rises exponentially (A). Blood ALA concentrations show a more complex relationship which is probably related lo changes in ihe urinary eserelion of ALA (R). The 

mathematical forms of Ihcse'rclationship*are 

Urinary ALA .OSfle*"" — "•* »04 l 

Blood ALA . J.2e-"» - 4l e " • • 4.9 

~.vv,„<rf^,a»-*-»vs»v*-i'~;«-. 

Lead and Heme Biosvnlhesis 99 

BLOOO 

A t « 

2 « 

MOO D LEAD (ll* . 

measured by methods similar to those used for urine (Haeger-Aronsen, 

1960). The mathematical association between urinary ALA and blood ALA 

takes Ihe form of a power curve. However in industrial exposure the relationship between blood AI.A and blood lead is an exponential one: hlood 

ALA concentration tends to plateau when the blood lead concentration is in 

excess or 3 pmol/l (Meredith et al., 1978) (Fig. I0B). 

C. Urinary Coproporphyrin 

Urinary coproporphyrin requires more complex quantitation techniques 

inherently less reliable than those for .-aminolevulinic acid: consequently, 

coproporphyrin in urine is generally a less satisfactory method of defining 

i;1 

•«v s;i-*££w?'f'ff 

;&tf£%YY 

WWm 

TJ 

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

100 Lead Toxicity 

excessive lead exposure. Like {-aminolevulinic acid, changes in the urinary 

coproporphyrin excretion start to take place only at greater concentrations 

of blood lead than those found in the general population and are therefore 

commonly used only in industrial lead exposure analysis. The normal values 

for urinary coproporphyrin and typical values found in a group or lead 

worker'; arc shown in Table 2. 

,.-

I). Dl'iod Protoporphyrin 

The use of blood protoporphyrin as an index of lead exposure has received 

considerable attention in the past few years. F.xamination of this index has 

used classical extraction procedures (Alessio et al., 1976) and Ihe development ol microfluoriiuctric methods with whole, hlood (Knmholtz ct al.. 

1972). Finally, the development of a portable front-face spcctrnlluorinicter 

has the advantage of rapidity of analysis and minimal preparation nl 

material, ll shows great promise in Tututc use for examination of load 

workers (Blumhcrg ct al., 1977). Measurement of protoporphyrin in blood 

as an index or lead exposure is limited by the very high incidence of iron 

deficiency anemia, which will also raise the concentration of protoporphyrin 

in blond. Such objections have in part been answered by consideration of the 

presence and absence of the zinc complex of protoporphyrin, which is more 

coniptonly found in lead exposure of long standing. It is irue to say, 

however, that in any disease of long standing in which protoporphyrin is 

raised, there arc increased concentrations of this zinc chelate. The highest 

concentrations of protoporphyrins of all in the blood will be found in subjects with erythropoietic protoporphyria in whom the primary defect is in 

the activity of the enzyme ferrochelatase in the bone marrow. Normal 

values for blood protoporphyrin and values commonly found in lead 

workers are shown in Table 2. Because the association between 

protoporphyrin in the blood and blood lead shows a positive exponential 

relationship (Fig. II), this method is of little use in examining environmental lead exposure, where a very high incidence of false positives will be 

found (Meredith ctal., 1979). 

F. Erythrocyte Dclta-aminoletulinlc Acid Dehydratase 

As wilh all enzyme measurements, the activily arrived at can vary 

considerably depending upon the methods used to quantitate it. This 

enzyme has benefited considerably from the development of a simple standardized method for the liuropean community (Berlin and Schallcr, 1974), 

although other methods for measurement of activily are equally acceptable. 

All depend ultimately upon the measurement of activity around the pH 

optimum of 7.4 of the rates of formation of porphobilinogen from 4-

aminolevulinic acid, usually in a phosphate buffer. Substances such as 

4 

BLOOD 

PROTOPORPHYRIN 

|>imol EPP/1'| 

Lead and Heme Biosynthesis 101 

.«.« • . ° . 1 * 

0 1 2 3 4 S 

BLOOD Pb (iimolfl) 

Flrore II . The relationship between blood lead concentrations and bio,*) protoporphyrin 

concentrations. Blood protoporphyrin concentrations rise exponentially, as blood lead 

concentrations rise. The mathematical relationship is 

Blood protoporphyrin - n.l4e,n,~"'"" *0.4S 

alcohol (Moore et al.. 1971) or zinc (Meredith and Moore. 1978) can 

interfere with the activity of this enzyme, but in general lead has the 

greatest effect or all upon the activity of this enzyme. The association 

between the activity of the enzyme and blood lead concentrations has been 

shown many times to have a negative exponential relationship (Fig. 5). This 

means that measurement of the activity of the enzyme is a particularly good 

biological index or lead exposure at environmental concentrations, i.e.. 

below 2 MM -
—, .*-. —-.-, :JV> v - -

• • -^^.-s : -, .»**,i^W'"» r .*.-»vs*- T. i-v- ^ 

F. Intercomparison of the Relative Value of 

Heme Biosynthetic Parameters in Assessment of Lead Exposure 

It is well established that all the heme biosynthetic parameters we have 

considered are highly significantly correlated with blood lead levels. A high 

coefficient does not, however, necessarily imply a high predictive validity. 

Such validity can only be established by the determination of the extent to 

which subjects are correctly classified as to their blood lead levels on the 

basis of the heme biosynthetic parameter under consideration. An evaluation of this type has recently been carried out (Meredith. Moore, and 

Goldberg 1979) on the basis of false positive and false negative results al two 

blood lead levels. The two blood lead levels selected were 1.75 fimol/l and 3.0 

fitnol/l, the first being the now generally accepted desirable upper limit for 

subjects with no known lead exposure and the second being representalive of 

a hlood lead value that could be encountered in industrial lead exposure. -For 

each of these two blood lead values, two values each were considered for urinary and blood ALA, urinary COPRO, blood protoporphyrin and erythrocyte ALA.D. These values were selected arbitrarily as belli}', those must 

ni.cly lo result in high predictive validity. Predictive validity was assessed in 

terms of percentage of false negatives, that is, a failure to detect subjects with 

raised blood lead levels, and percentage of false positives, that is. a false prediction of raised blood lead levels. Results of this analysis in a group of subjects with known industrial lead exposure and subjects with no known 

excessive lead exposure arc shown in Tabic i. It is clear from this table that 

Table 3. The predictivevalidity of heme hjosynlheticparanicters in.the assessment., 

of elevated blood lead concentrations 

Blood ALA 

(<imol/l) 

Urinary ALA 

(mmol/mol creatinine) 

Urinary coproporphyrin 

(iimol/mol creatinine) 

Erythrocyte ALA.D 

(nmol ALA/min/ml 

rbc 

Blood protoporphyrin 

(«moll-.PP/l) 

> J 5 

>J.O 

>2.5 

>2.0 

> 12.5 

> in.o 

<20 

<25 

>I.O 

>0.75 

Blood lead Gimol/I) 

Environmental 

OI.75 ) 

False 

positive 

Cr) 

5.4 

18.6 

4.8 

11.4 

10.2 

15 6 

3.7 

6.3 

69 

20.6 

False 

negative 

ro 

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 

Industrial 

O3.0 ) 

False 

positive 

CO 

1.2 

66 

1.2 

4.2 

JO 

11.4 

1.6 

9:0 

2.6 

9.0 

False 

negative 

('") 

4.X 

1.8 

.1.6 

J.O 

7.: 

3.6 

4.8 

OS 

9.0 

4.2 

only erythrocyte ALA.D offers a good index of environmental lead exposure: 

that is. when the number of raise negative results is reduced to an acceptable 

level {i.e., less than 5%) by reduction or the threshold value, the number of 

false positives remains at an acceptable level (i.e., less than 10%). At the 

higher blood lead level it is apparent that, with the exception of urinary 

COPRO. all the parameters are acceptable screening tests on the basis or predictive validity. 

Thus, for the screening of a broad population. Ihe activity of erythrocyte ALA.D is a highly sensitive index of both environmental and 

industrial lead exposure. However, due to the relative rapidity and simplicity of blood protoporphyrin determination using the "hcmatofluorimeter," it is probably that the latter test is more expedient Tor the screeninn of an industrially exposed group of workers. 

G. Hemoglobin 

It is only in clear cases of lead poisoning that hemoglobin levels start to rail. 

In general, this will only happen when blood lead levels arc in excess or 4 

jiMYsO jtg/100 ml). As shown before. AI.A synthase activity is inversely 

related to hemoglobin and hemoprotcin concentrations during lead exposure 

(Campbell et al'., 1977; Goldberg et al.. 1978); other studies have shown a 

similar association with blood protoporphyrin levels (Meredith ct al.. 1979). 

A rise in blood protoporphyrin lc\els will reflect depression or heme production more accurately than will depression or erythrocyte ALA dehydratase. 

This is consistent with the previous suggestion that there are critical tissue 

lead concentrations oT about 2 |iM (4O»rg/IO0 ml): This corresponds to~ 

erythrocyte protoporphyrin levels or about 0.75 mM and erythrocyte ALA 

dehydratase activity or 20 nmol ALA utilized/min/ml red blood cells. At 

this concentration or lead, the inhibition or heme synthesis is sufficiently 

great to depress the synthesized free heme and therefore hemoglobin and 

thus to induce ALA synthase activity. 

IX. CHANGES IN DRUG METABOLISM Dl'RING LEAD EXPOSURE 

A large proportion or heme synthesized in the liver acts as the prosthetic 

group or the hemoprotcin cytochrome P-450. This hemoprolein is the terminal oxidase or the microsomal mixed fitnetion oxidase system that is 

involved in the metabolism or many drugs. The possibility that drug 

metabolism might be altered in lead intoxication due lo depressed synthesis 

or microsomal cytochrome P-450 has been considered in both man and animals. 

II •• 

'*fJ 'rti^i •• • ,v 

'Ml ' 
• r»f >**.•:>.-.»»:-•«. 1.»?.ss*— .-••- ir-— 

A. Animal Studies 

Initial studies in animals produced contradictory results. The work o l 

Ribeiro (1970) on the effects of arsenic, beryllium, lead and mercury on 

mouse liver drug-metabolizing enzymes indicated that pretreatment or mice 

with lead nitrate to give liver levels or 10' ' and 10"* M Tailed to alter 

hexobarhital sleeping times or certain microsomal enzyme activities that 

were measured in vitro. When lead was added to control microsomes at 

concentrations between 10 ' and 10"' M , no inhibition or hexobarhital oxidase was noted. In contrast, the results of a number of investigators 

(Alvares et al.. 1972: Scoppa et al., 1973: Lgan and Cornish. 1973: Chow 

and Cornish. 1978) suggest that lead has a significant effect on microsomal 

drug metabolism. Alvares et al. (1972) found a 40 to 50% decrease in 

metabolism by hydroxylation and dcmcthylation and a significant decrease 

in cytochrome P-450 levels and hc.xoharbital sleeping times 24 hours after ' 

the intravenous injection of lead chloride. From the results of their studies, 

the authors considered that lead exerts its effect on drug metabolism via an 

inhibition or heme synthesis. 

Scnppa et al. (1973) attempted to verify this hypothesis hy correlating 

the inhibition of hlood and liver ALA dehydratase by lead with drug 

metabolism. They were able to demonstrate an association between lowered 

activity of this enzyme and depression in cytochrome P-450 levels and an 

associ itcd depression af in vitro activities or Ihe microsomal mixed function 

oxidase system. 

These studies did not, however, provide unequivocal evidence of the 

existence or a relationship between depressed heme synthesis and altered 

drug metabolism. Goldberg et al. (1978) provided farther evidence that lead 

has a profound effect upon the microsomal mixed function oxidase system. 

An increased dose of lead to rats resulted in a progressive decrease in 

cytochrome P-450 content and a progressive decrease in the activities or 

aniline hydroxylase and aminopyrine demelhylase. As had been noted previously (Maxwell and Meyer, 1976). there was an increase in the activity oT 

the rate-limiting enzyme or heme biosynthesis. ALA synthase, associated 

with the impairment of the mixed function oxidase system. An inverse relationship between AL A synthase and cytochrome P-450 content was" 

observed: this is illustrated in Figure 12. This inverse relationship provides 

farther evidence or the control mechanism whereby a decrease in the regulatory pool or heme, as assessed by the microsomal content of the hemoprotcin cytochrome P-450. results in an increase in the activity or ALA 

synthase by a negative Tecdback mechanism. 

From in vitro studies (Scoppa et al., 1973: Goldberg et al., 1978), it is 

evident that lead has no direct marked effect on the microsomal mixed function oxidase system. It is apparent that the depletion of cytochrome P-450 

125r 

too 

I 

I 

•sr s 

0- 8 

07S 

4) E O50r 

5 | 

U il 

2 ? 

?. I 0-25 

o — 

n l i t i i , 

0 5 0 100 150 20 0 

ALA. S (nmole At-A formed / 

9.homogenate protein/rs) 

2 5 0 

Figure 12. The changes in hepatic microsomal cytochrome P-450 and hepatic AI.A synthase 

in lead-lrealed rats. In this graph, the lead-trcalcd animal, :irc shown as closed squares and the 

untreated animals are shossn as closed circles. As lead concentration rises, so Ihe activity ol 

AI.A synthase rises; concurrently, there is an exponential decrease in Ihe concentration of 

hepatic microsomal cytochrome P-450. The mathematical form of this relationship is 

Cytochrome P-450 - • - 0.0911 

in lead intoxication in animals can be mediated through two possible 

mechanisms: the first is associated with a reduction in the activity of the 

intermediate enzymes or heme biosynthesis ALA dehydratase. COPRO oxidase and ferrochelatase synthesis (Goldberg et al.. 1978). The second possible mechanism is associated with an increase in heme degradation due to 

. increased activity of the heme degradative enzyme heme oxygenase (Maines 

and Kappas, 1976b; Goldberg ct al., 1978). This latter mechanism conflicts 

with a previous model suggesting thai ALA synthase and heme oxygenase 

are interrelated with respect to their regulation by heme Bissell and Hammaker (1976). They further predicted that these enzyme activities vary 

reciprocally. This theory has experimental support from the studies of 

Schacter (1975). From this theory, one would expect depletion ofthe regulatory heme pool, which would activate ALA.S but depress heme oxygenase. 

Experimental evidence docs not support this theory. Lead pretreatment of 
r v , ', ' ..;•*«**. ...M—I—•' * •••^*-* 

il.ld I'.A.vll V 

rats results in inhibition or hepatic heme synthesis and depletion or the regulatory heme pool. Thus there is not only an associated increase in the 

activity or ALA synthase, but also a rise in the activity or heme oxygenase. 

These findings are, however, consistent with the theory postulated by 

Maines and Kappas (1976c) that the mode of action of metals in inducing 

heme oxygenase is based on a repressor component in the regulatory 

mechanism or heme oxygenase. They suggested that this repressor has an 

-SH active constituent, the oxidation-reduction capacity ol which is 

necessary for controlling heme oxygenase production. If the oxidationreduction cycle of Ihe -SM groups of this component are blocked, as would 

occur after treatment wilh lead, which has a high affinity for -SH groups, 

the regulatory function of this cellular constituent on heme oxygenase would 

be lost. The system would then function without regressive regulation, leading to an exaggerated synthesis or the enzyme. 

Thus, the impairment or the mixed function oxidases demonstrated in 

lead treated animals has clearly been.'shown to be mediated throur.h a 

decrease in heme biosynthesis und an increase in heme degradation. As yet. 

il is not possible to conclude which of these two mechanisms is the more 

important in the reduction or microsomal P-450 content; both mechanisms 

arc possible and they are not mutually exclusive. 

ft. Human Studies 

The attention focused on the effects or lead on in vitro drug metabolism in 

animals has resulted in a number or investigations into the effects or lead 

exposure in man on in vivo drug metabolism rates. A preliminary study was 

carried out by Alvares et al. (1973) in two children who showed clinical and 

biochemical manirestations or acute lead poisoning. As a measure or in vivo 

drug metabolism rates, phenazone (antipyrine) half-lives were determined 

both before and after chelation therapy. The determination of phenazone 

half-lire in man offers an index of drug metabolizing capacity because an 

oral dose is completely absorbed and is metabolized exclusively by the 

hepatic cytochrome P-450 dependent microsomal mixed runction oxidase 

system. The phenazone hall-lives in the two subjects were found to be significantly longer lhan in a group or normal children, while chelating therapy 

led to a reduction in half-lire in both subjects. The same authors (Alvares et 

al.. 1976) were, however, unable lo demonstrate a statistically sigrvficant 

change in the rate of elimination or phenazone from (he plasma or eight 

industrially exposed lead workers following chelation therapy. They did 

note that in seven of the eight subjects there was a reduction in phenazone 

half-life following chelation therapy. 

Subsequent studies have demonstrated that in acute lead intoxication in 

man there is a depression in the rate or drug metabolism (Meredith et al., 

1977; Fischbcin et al.. 1977). In the first or these two studies (Meredith et 

al.. 1977), plasma phenazone elimination rates were determined in a group 

or 10 demolition workers admitted to hospital with clinical and biochemical 

symptoms or excessive lead exposure before, immediately after and again at 

least 12 weeks after the cessation of chelation therapy. At the same time, 

erythrocyte ALA dehydratase activity, blood lead levels and hemoglobin 

levels were determined. Chelation therapy was found to be associated with a 

highly significant (p < 0.005; paired t analysis) decrease in phenazone hairlire and an increase in clearance bnih immediately after therapy and again 

when measured at least \2 weeks alter chelation therapy (Fig. 13). The 

changes in phenazone elimination rates following chelation therapy were 

also associated with decreased blood lead levels and increases in both hemoglobin levels and erythrocyte ALA dehydratase activity. The increase in 

phenazone elimination rates in (lie subjects following chelation therapy was 

significantly correlated with the fall in blood lead levels (p < 0.005). 

In a similar study on five subjects with biochemical manifestations of 

chronic lead intoxication. Fischbcin et al. (1977) noted a slight but statistically significant decrease in phenazone half-lifo following chelation 

therapy. However, (he rats- or elimination or phenylbutazone was unaffected 

by chelation therapy. 

It is apparent that Ihe acute administration or lead to rats is associated 

wilh decreases in cytochrome P-450 contenl or hepatic microsomes. The 

increase in in vivo drug metabolism rales measured following chelation 

therapy or lead-intoxicated human subjects (Meredith el al.. 1977: Fischbcin 

et al., 1977) is probably due to depressed levels or the hepatic microsomal 

hemoproteiri cytochrome l';450." 

X. THE ANEMIA OF LEAD POISONING 

Three mechanisms are involved in the development or anemia during lead 

poisoning. Firstly, there is depression or heme biosynthesis; secondly, there 

is disturbance or globin synthesis; and. finally, there is hemolysis or red 

blood cells. The development or such anemia will occur at two times, during 

red blood cell formation and during red blood cell destruction. Since lead is 

preforcntially deposited into bone (Fig. 4), it is clear that during red cell 

formation in the bone marrow the concentrations or lead in the marrow will 

be very much greater than that in the blood (Albaharry. 1972). In these circumstances, the developing cells within Ihe bone marrow will be particularly 

affected by lead. Therefore one would expect dcrcctive erythropoiesis 

(Landow et al., 1973). Ilcrk et al. (1970) suggested that erythropoiesis is 

completely effective during lead intoxication in a human adult with lead 

poisoning. However, Dagg et al. (1965), in a study of two patients with lead 

$m &*$&%'•''•••>{' 

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Hi tf^j^s-r 
24-1 

preEDTA 

immediately 

post-EDTA 

> 12 weeks 

post-EDTA 

Figure 13. The efTccts of chelation therapy on phenazone half-life in lead-poisoned workers. 

The rate of phenazone metabolism was examined in 10 lead workers at the lime ol lead poisoning, immediately after ethylene diamino tetra-acetic acid (EDTA) chelation therapy and more 

than 12 weeks after chelation Iherapy. In all cases, the rale of phenazone metabolism rose 

following chelation of lead from the systems of these lead-poisoned subjects, as evidenced by a 

decrease in ihe phenazone half-life. This could not be attributed lo existing concentrations of 

EDTA. 

poisoning before and after treatment with penicillamine, showed that the 

anemia of lead poisoning is due partly to hemolysis and partly to inhibition 

or iron utilization. 

Lead affects protein synthesis: specifically, it will inhibit the synthesis 

or globin. It interferes with amino acid incorporation and. when reticulocytes are incubated with lead, one finds disaggregation of polyribosomes. 

In vitro experiments have shown that, in addition to this decrease in globin 

synthesis, the rate or production or alpha and beta chains become out or 

phase, with suppression or alpha chain production being more marked than 

that or the beta chains. In vivo, there appears to be some compensation Tor 

this to restore the balance or synthesis: the significance or this finding is in 

some doubt (White and Harvey. 1972). The final effect that lead may have 

on the red cell is its effect on the red cell membrane. The normal life span or 

the red cell is about 120 da\s. In hemolytic anemia, the mean life span or 

these cells measured by radioisotope techniques is reduced (Griggs and Harris, 1958; Waldron, 1966). This shortening or the red cell life span is 

probably due to direct toxic effect upon the cell membrane. 

Lead interferes with the Na'-K* dependent ATPasc activity in the red 

cell (Hasan et al., 1967): as a consequence, red cells incubated with high 

concentrations of lead leak large quantities or K'. This loss is irreversible 

and is thought to be caused by interference with this energy-requiring pump. 

At the same time, there is little change in the input or Na' into the cell; in 

consequence there is a rise in Na' concentration in lead poisoned erythrocytes, associated with potassium-mcdialcd shrinkage or the cell. This K' 

leakage almost certainly leads to an increased mechanical fragility or the 

red blood cell and consequent lysis. The importance 6r anemia in lead 

poisoning is readily seen in the incidence or the various features or lead 

poisoning shown in Figure 1-1, where about 90% or lead-poisoned workers 

were found to have some degree or anemia. As the degree or lead poisoning 

increases, the hemoglobin concentration in the blood decreases. At the same 

time, there are rises in the activity or .-aminolevulinic acid synthase, presumably in an attempt lo compensate for the decreased concentrations or 

free heme within the cell. The type of anemia found in these workers is 

usually a normocytic. normochromic anemia. In affected children, it may 

become macrocytic and hypochromic anemia. 

XI. A COMPARISON OF 

LEAD POISONING AND ACUTE HEPATIC PORPHYRIA 

A. General Considerations 

Although acute porphyria and lead poisoning are conditions of different 

etiology, one being genetically determined and the second acquired, il is or 

interest to compare the similarities or their clinical features and of the excretion patterns or porphyrin precursors and porphyrins. These similarities 

often reflect similarities in the abnormalities or enzyme activity and 

porphyrin, metabolism (Dagg et al., 1965: Gibson and Goldberg, 1970; 

Campbell et al., 1977; Doss and Tiepermann. 1977). The features of acute 
lU2-fQlifiBUfi Arun IMTHWITiiai^csrnijua 

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futlnlAu.cidiAHi 

liqure 14. The incidence of the typical features of lead poisnninit and or acute intermittent 

p'rphvria. The incidence of the effect of lead poisoning in 10 industrially exposed subjects, all 

of sshom had hlood lead concentrations excecdins; 4 wmol/l. is demonstrated in this histogram, 

liifttncr wilh the simii-fr incidence of features of acute intermittent porphyria in 50 subjects 

iillt) this disease. Sortie features ar* (Sen only in lead poisoning, such as hyperuriomia. 

metallic tjstc and. in some cases where poor dental hygu-nce co-exists wilh lead puisonine. ire 

gingival blue line. 

porphyria, which are abdominal pain, constipation; vomiting, paralysis or 

paresis, are similar to those found in lead intoxication and. indeed, the Incidence of these in both conditions is also similar (Fig. 14). In lead poisoning, 

the paralysis or paresis is entirely of the motor neuron type, while in acute 

intermittent porphyria 10% or the subjects show upper motor ncur'ori 

involvement and organic brain disorder (Goldberg. 1968). 

In. terms or excretion or either porphyrins or their precursors, the 

overall pattern found in lead poisoning is in no way identical to any or the 

porphyrias, although some similarities exist. In all acute porphyrias there is 

excessive excretion or {-aminolevulinic acid; the same is found in lead 

poisoning. In terms or porphyrin excretion, none or the porphyrias produce 

a pattern similar to lead poisoning: exceptions are Ihe urinary excretion or 

coproporphyrin, which is found in hereditary coproporphyria, and the 

excessive production or protoporphyrin seen in varicgalc porphyria and 

erythropoietic protoporphyria. A comparison or the patterns of change of 

enzyme activity in lead poisoning and in acute intermittent porphyria and 

hereditary coproporphyria shows the reason for these differences (Fig. 15). 

In acute intermittent porphyria, there is depression or porphobilinogen 

deaminase activity leading to increased activity or ALA synthase, while in 

hereditary coproporphyria there is depression or coproporphyrinogen oxidase activity with subsequent increases in the activity or ALA synthase. In 

lead poisoning, three enzymes are clearly inhibited (ALA dehydratase, 

coproporphyrinogen oxidase and ferrochelatase) with consequent elevation 

in 6-aminolevulinic acid synthase. 

There is thus one feature common to each of these porphyrias and lead 

poisoning; thai is a rise in the activity of AI.A synthase, the only other feature or two of Ihe aculc porphyrias that might be found in lead poisoning is 

a porphyrin-mediated solar photosensitizalion of the skin. In variegate 

porphyria, increased protoporphyrin leads to photosensitivity; in a proportion of cases with hereditary coproporphyria, a photosensitive skin eruption 

may he found, although this is never the case in acnlc intermittent 

porphyria. Pholoscnsitization in lead poisoning is uncommon, but it has 

AI.A.S AI.A.D UPO.S URO.D f.OPRO.0 FEPPO.C. 

* t ' , LEAD 

POISONING 

ACI'IL 

; iruEWintEnT 

. PORPHYRIA 

HEREDITARY 

COPROPORPHYRIA 

* 

* 

4 

iFotttM^+ioimt 

• I 

• • | INCREASE oo DECREASE »B^ S|> ) IN ENZYME Activity 

0 - NO CHANGE IN ENZYHE ACTIVITY,KITH RESPECT TO NOIWAL VALUES 

ALA.S -IAMINOLAEVULINIC ACID SYNTHASE 

ALA.D -HAMINOLAEVULINIC ACID DEHYDRATASE 

URO.S - UROPORPHYRINOGEN 1 SYNTHASE 

URO.D - UROPORPHYRINOGEN DECARBOXYLASE 

COPRO.0 - COPROPORPHYRINOGEN OXIDASE 

FERRO.C - rtRROCHtLATASE 

Kipire 15. A comparison of the changes in enzyme activity in lead poisoning, acute intermittent purphyria and hereditary coproporphyria. Although there are many similarities in Ihe excretion of intermediates of Ihe heme hinsvnlhetie pathway in lead poisoning and these two acute 

porphyrias, the only truly common point to all is Ihe rise in activity of 4-aminnlcvulinic acid 

synthase. There are also similarities in depression of coproporphyrinogen oxidase in both lead 

poisoning and hereditary coproporphyria. 

1.1 

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been reported (Allan et al., 1975). Various reasons have been advanced for 

the difference in photosensitizing lesions in lead poisoning and the 

cutaneous porphyrias, such as the presence of zinc chelates or protoporphyrin in lead poisoning (Lamola et al., 1975) and altered plasma binding ol porphyrins (Piomelli et al.. 1975). It is possible that depression or 

enzyme activity at an earlier stage in heme biosynthesis in lead poisoning 

than in hereditary coproporphyria means that lesser quantities or substrate 

arc being metabolized by the depressed coproporphyrinogen oxidase. In 

consequence, lesser quantities or coproporphyrin are able to build up in the 

circulation. The defect may also lie in oxidation-reduction factors, since 

within the body Ihe porphyrin normally exists as the reduced porphyrinogen 

which is only photoactive when oxidized to the porphyrin form (l-ig. 2). This 

view is supported by the feature or altered terminal oxidation during lead 

exposure. ',. 

The liver is the main site or disturbance or porphyrin metabolism in 

acute porphyria. When ferrochelatase activity in Ihe bone marrow is 

depressed by lead, however, hepatic cells show a compensatory increase in 

the activily or this enzyme (Otrzonsek, 1967). This may explain some or the 

clinical differences between lead poisoning and acute porphyria, in which 

the principal effects of acute porphyria are in Ihe liver, whereas those of 

lead poisoning would appear to be in ihe bone marrow. 

R. The Influence or dclta-aminolemlinic Acid 

The role or the heme biosynthetic precursor ALA within the body has been 

Ihe subject or several investigations, owing to its involvement in the 

pathogenesis or lead poisoning, acute porphyria and hereditary tyrosinanemia (Becker and Kramer. 1977). Attacks or acute porphyria are 

characterized, among other signs, by ascending paralysis, which can 

progress to quadriplcgia and to respiratory failure. During these attacks, the 

blood and urinary levels or ALA reach very high values. In lead intoxication, blood ALA concentrations may double or more (Meredith et al.. 1978) 

and hepatic concentrations or lead in these circumstances have been found 

to be increased fourfold (Sukcta et al., 1975). In normal circumstances, 

blood ALA concentrations are in the region or 3 >imol/l (Meredith et at., 

1978). During attacks ol acute porphyria, the concentration or ALA in 

plasma has been reported to be as high as 24 /ig/ml (200 pmol/l). at which 

lime the ALA concentration in cerebrospinal fluid was 2.8 pg/ml (23 

/itnol/l) (Sweeney ct al., 1970). These concentrations are commonly much 

lower (Moore and Meredith, 1976: Percy and Shanley, 1977). 

In laboratory animals, high levels of ALA in Ihe body arc associated 

with various behavioral changes, including some impairment of voluntary 

movement. This effect has been observed after direct administration of 

ALA (Moore and Meredith. 1976: Shanley el al.. 1975; Yuwiler et al., 

1970). ALA has also been shown to have various pharmacological activities 

(McUillion et al., 1975: Loots et al.. 1975: Becker et al.. 1976) and indeed to 

be taken up by various tissues including the brain (Becker et al., 1974; 

Moore and Meredith, 1976: Shanley ct al.. 1975). Some workers, however, 

have suggested that there are few behavioral effects from ALA in animals 

(Shanley et al., 1975) or in man (Doss. 1976) or loxic effects in mice (Kennedy el al., 1976) or in rats (Picrach et al., 1977). 

In other experiments, effects or {-aminolevulinic acid (ALA) on social 

behavior were examined by elholupical analysis t>r encounters between male 

mice. Thirty to forty minutes after receiving an injection of ALA, mice 

explored the cage and scanned less frequently than saline-injected controls 

and showed longer periods of immobility (Cutler et al., 1979b). Blockage or 

neuromuscular runction appears to be the predominant effect or AI.A in 

those preparations where the muscle becomes unresponsive to nerve 

stimulation. A separate toxic action, possibly affecting membrane transport 

or muscle metabolism, appears to predominate when inhibition or muscular 

contraction precedes blockage of the action potenlial, at which time the 

muscle becomes temporarily unresponsive lo direct electrical stimulation. 

Some of the known actions or ALA that may be responsible lor its 

effect on nerve-muscle function include an inhibition of membrane sodium 

transport (Kales el al.. 1971) and an inhibition or Na'-K ' dependent 

ATPase (Becker el al.. 1971) wilh associated increase in capillary 

permeability (McCiillion et al.. 1975). Becker ct al. (1975) found that ALA 

significantly reduced the resting membrane potential or frog sartorius 

muscle; this is an effect which would be expected from an inhibition of Na*-

K* dependent ATPase. However, Feldman et al. (1968). using the rat 

phrenic nerve hemidiaphragm preparation, obtained results suggestive of 

hyperpolarization. They found that ALA in amounts that could be expected 

in the acute phase or porphyria produced a presynaptic inhibition or the 

potassium-augmented miniature endplate potential frequency and appeared 

lo reduce membrane excitability. It is possible that ALA may inhibit the 

transport or sodium in both directions across the cell membrane by blocking 

the channels through which sodium is transported into the cell during 

propagation or an impulse, in addition to its effects on ATPase. Some 

interference with ion transport across the cell membrane was indicated in 

the experiments or Cutler ct al. (1979a) by Ihe presence or polyphasic action 

potentials thai occurred in frog nerve-muscle preparations following 

application or ALA. 

Inhibition or Na'-K" dependent ATPase may also affect neuromuscular fonction. Many different experimental conditions known to inhibit this 

enzyme deplete acetylcholine concentrations on presynaptic sites (Vizi, 

i I 

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1977). Such an effect would provide an explanation for the ALA-induced 

spontaneous contractions of muscle and for the initial increase in muscle 

contraction prior to blockage. 

A further study of the mode of action or ALA is likely to provide some 

interesting information that may shed light on the role or AI.A in lead 

poisoning. Such studies do not exclude the possibility or effects from the 

metabolites or ALA. the monopyrroles such as porphobilinogen, which have 

also been shown to cause presynaptic neuromuscular inhibition (Feldman et 

al.. 1971). 

These various studies have clearly demonstrated that the interaction or 

lead with two enzymes or heme biosynthesis results in marked increases in 

both urinary and hlood levels or AI.A. This passes through the blood-brain 

harrier and can cause marked changes in spontaneous activity or rodents. 

The possibility therefore exists that ALA can act cither directly or indirectly 

on neuromuscular function and cause some or all or the neurological and 

behavioral effects of lead toxicity. 

XII. SUMMARY AM) CONCLUSIONS 

The influence of lead upon heme biosynthesis and upon the initial stages of 

heme degradation has been reviewed. The principal effects or lead arc upon 

the biosynthetic enzymes. The enzymes inhibited are {-aminolevulinic acid 

dehydratase, coproporphyrinogen oxidase and ferrochelatase. The greatest 

diminution of activity is in the first of these, {-aminolevulinic acid 

dehydratase. As a consequence or depressed synthesis or heme, the activity 

~ of the initial and rate controlling enzyme or the pathway, {-aminolevulinic 

acid synthase, is increased, as is the activity or heme-oxygenase, the first 

enzyme in the biodegradative sequence. 

As a consequence or these changes in enzyme activity there is an 

increase in the production and excretion or the intermediates or the 

biosynthetic pathway, {-aminolevulinic acid, coproporphyrin and 

protoporphyrin. The changes in these intermediates have been used together 

with {-aminolevulinic acid dehydratase as secondary indices of lead 

exposure.Since synthesis of heme is depressed, so also is the synthesis of> 

hemoproteins such as cytochrome P-450 and hemoglobin. Depression of 

cytochrome P-450 by lead will cause alterations in hepatic drug metabolism 

in both animals and man. 

Anemia will also develop during lead poisoning due to three factors. 

These are depressed heme synthesis, altered globin synthesis and red cell 

fragility resulting in hemolysis. 

Finally, because lead poisoning is so similar in presentation to the 

acute porphyrias, these are compared and contrasted. It is considered possible that the associated neurological manifestations or these diseases occur in 

part through the overproduction or the porphyrin precursor {-aminolevulinic acid, which is shown to be neuroactive. 

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