Erythrocyte zinc-protoporphyrin

An alternate mechanism to explain the etiology of elevated erythrocyte ZPP during lead exposure has its basis in the relative affinities of iron and zinc as substrates for ferrochelatase. Ferrous iron is the preferential substrate of ferrochelatase and is also an effective inhibitor of zinc utilization by this enzyme. However, when the concentration of iron as Fe decreases to suboptimal levels, as in iron deficiency, zinc is utilized by ferrochelatase as a substrate. Studies have suggested that lead decreases the availability of Fe as a substrate for ferrochelatase by inhibiting the enzymatic reduction of Fe to Fe within mitochondria (Taketani et al. 1985), as required for use 

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PbB concentrations reflect the absorbed dose of lead. However, the interpretation of PbB data depends on a knowledge of the past history of exposure to lead. This is because in the body, bone constitutes the major lead sink and this results in lead having a long body half-life. Thus, in the absence of intense exposure to lead for a considerable period up to its body half-life, the PbB concentrations reflect recent lead exposures. However, if intermittent exposure to lead is occurring in several distinct environments, the PbB concentration reflects both recent and past exposures to lead. Thus, biological effects for populations with the same PbB concentrations may not be the same since different exposure times scales may be involved. This is the reason why free erythrocyte protoporphyrin (FEP) and erythrocyte zinc protoporphyrin (ZPP) have been used as additional biological markers since their elevation is more related to chronic lead exposure than acute lead exposure (see Section 2.7). 

Odone P, Castoldi MR, Guercilena S, et al. 1979. Erythrocyte zinc protoporphyrin as an indicator of the biological effect of lead in adults and children. In International Conference on Management and Control of Heavy Metals in the Environment, London, United Kingdom, September. Edinburgh, UK CEP Consultants, Ltd., 66-69. 

Zinc protoporphyrin IX is a normal metabolite that is formed in trace amounts during haem biosynthesis. However, in iron deficiency or in impaired iron utilization, zinc becomes an alternative substrate for ferrochelatase and elevated levels of zinc protoporphyrin IX, which has a known low affinity for oxygen, are formed. This zinc-for-iron substitution is one of the first biochemical responses to iron depletion, and erythrocyte zinc protoporphyrin is therefore a very sensitive index of bone-marrow iron status (Labbe et ah, 1999). In addition, zinc protoporphyrin may regulate haem catabolism by acting as a competitive inhibitor of haem oxygenase, the key enzyme of the haem degradation pathway. However, it has been reported... 

Determination of Erythrocyte Zinc Protoporphyrin Methodological Problems... 

Elevated erythrocyte zinc protoporphyrin indicates iron-deficient erythropoiesis. Protoporphyrin concentrations may also be elevated by inflammation and lead exposure. 

Zinc protoporphyrin in blood After 1 month exposure 2 50 pg/100 ml erythrocytes or lOOpg/IOOmI blood B... 

Inhibition of ferrochelatase in the heme pathway causes accumulation of protoporphyrin in erythrocytes (CDC 1985). Most protoporphyrin in erythrocytes (about 90%) exists as zinc protoporphyrin (ZnPP). This fraction is preferentially measured by hematofluorometers. Extraction methods measure all the protoporphyrin present, but strip the zinc from the ZnPP during the extraction process. For this reason,... 

Chisolm JJ Jr, Brown DH. 1979. Micromethod for zinc protoporphyrin in erythrocytes Including new data on the absorptivity of zinc protoporphyrin and new observation in neonates and sickle cell disease. Biochem Med 22 214-237. 

Chromatographic determination of protoporphyrins in erythrocytes has the same indications as the spectrophotometric one. In addition, the method enables the differentiation between zinc-protoporphyrin and (metal-)free protoporphyrin. The first is elevated in iron deficiency and lead intoxication, the second in erythropoietic protoporphyria. 

Each laboratory should establish its own reference values. Those given below can only be interpreted as a guide. Free protoporphyrins in erythrocytes are rarely detectable in healthy individuals. Zinc protoporphyrin <1.3 pmol/1 (mean 2SD), free protoporphyrin < 0.2 pmol/1 (detection limit). 

The insertion of ferrous iron into the porphyrin ring in the biosynthesis of heme is catalyzed by the enzyme ferrochelatase. A deficiency in ferrochelatase activity results in an accumulation or the excretion of unchelated protoporphyrin in patients with erythrohepatic protoporphyria. Ferrochelatase catalyzes the synthesis of a range of metalloporphyrins,628 and, for example, produces zinc protoporphyrin in erythrocytes of patients with iron-deficiency anaemia. 

FECH (also known as heme synthase) is an iron-sulfur protein located in the inner mitochondrial membrane. This enzyme inserts ferrous iron into protoporphyrin to form heme During this process, two hydrogens are displaced from the ring nitrogens. Other metals in the divalent state will also act as substrate, yielding the corresponding chelate (e.g., incorporation of Zn into protoporphyrin to yield zinc protoporphyrin). In iron-deficient states Zn successfully competes with Fe in developing red cells so that the concentration of zinc protoporphyrin in erythrocytes increases. Furthermore, other dicarboxylic porphyrins will also serve as substrates (e.g., mesoporphyrin). 

Hart D, Piomelli S. Simultaneous quantitation of zinc protoporphyrin and free protoporphyrin in erythrocytes by acetone extraction. Clin Chem 1981 27 220-2. 

Blood lead levels provide the best indicators of lead poisoning but do not reflect total body burden (Lee and Moore 1990). The inhibition of erythrocyte 8-aminolevulinic acid indicates lead exposure, but most centers still use blood lead levels for screening (Lee and Moore 1990 Roper et al. 1993 Schaffer and Campbell 1994). Zinc protoporphyrin indicates neurotoxicity from lead but does not have the sensitivity for assessing low levels of exposure (Anger and Johnson 1985 Royce and Needleman 1995). Radiological examination of the abdomen and long bones does not reliably portray exposure. The same holds true for the examination of red blood cells for basophilic stippling and the assay of hair and nail levels for lead (Roper et al. 1993). The Centers for Disease Control and Prevention (CDC) does not recommend use of scarification of the forearm with 25% sodium sulfite solution to assess for black discoloration of skin, a procedure recommended in some sources. Medical centers perform an edetate disodium calcium provocative chelation test with urinalysis and complete blood... 

Several other classes of proteins have also been implicated as possible targets for lead, including other proteins in the heme biosynthetic pathway, leadbinding proteins in the kidney and brain, and heat shock proteins (342, 500-502). Lead is known to affect several steps in the heme biosynthetic pathway other than that catalyzed by ALAD Other profound effects include stimulation of 5-aminolevulinic acid synthase (ALAS) and decreased levels of iron incorporation into protoporphyrin by ferrochelatase (see Section VI.E.2 and Fig. 34) (10, 503-505). However, not all of these effects are due to direct interactions between lead and enzymes in the heme biosynthetic pathway. For instance, the widespread assertion that lead inhibits ferrochelatase is not supported by studies on the isolated enzyme (506, 507). Furthermore, increased levels of both erythrocyte protoporphyrin IX (EP) and zinc protoporphyrin (ZPP) are observed at high BLLs, suggesting that ferrochelatase is stiU competent to insert zinc into EP and that the increased levels of EP and ZPP associated with lead poisoning are most likely caused by lead interfering with iron uptake or transport (see Sections VI.C.4 and VI.E) (10, 506, 507). 

B. Elevations in free erythrocyte protoporphyrin (FEP) or zinc protoporphyrin (ZPP) (> 35 mcg/dL) reflect lead-induced inhibition of heme synthesis. Because only actively forming and not mature erythrocytes are affected, elevations will typically lag lead exposure by a few weeks. A high blood lead in the presence of a normal FEP or ZPP therefore suggests very recent exposure. Protoporphyrin elevation is not specific for lead, and may also occur with iron deficiency. Protoporphyrin levels are not sensitive for low-level exposure (blood lead < 30 mcg/dL). 

The other indirect measures which can be used to monitor lead exposure are changes in the levels of a range of enzymes and metabolites involved in the synthesis and operation of haem. Thus, increases in the levels of free erythrocyte protoporphyrin (FEP) or of zinc protoporphyrin (ZPP) in blood can be associated with increased levels of lead in blood, as can decreased levels of activity of the enzyme delta-aminolaevulinic acid ddiydratase (ALAD). Similarly increases in the levels of urinary coproporphyrin (CP) and urinary aminolaevulinic acid (ALAU) also reflect increased lead exposure. These measures are not, however, always reliable since they can be affected by other factors, for exattqrle ZPP may be increased by iron deficiency. Measurements of these parameters tend, therefore, to be us only in conjunction with, and to provide supplementary data to, blood lead measurements. 

Another enzyme, ferrochelatase, is also inhibited at low blood lead levels. Inhibition of ferrochelatase leads to increased free erythrocyte protoporphyrin (FEP) in the blood which can then bind to zinc to yield zinc protoporphyrin. At a blood lead level of 50 [ig/dl or greater, nearly 100 percent of the population will have an increase in FEP. There is also an exponential relationship between blood lead levels greater than 40 [ig/dl and the associated ZPP level, which has led to the development of the ZPP screening test for lead exposure. 

Elucidation of some of the mechanisms of lead toxicity on the cellular and biochemical level has led to the development of several relatively sensitive biomarkers of lead exposure and toxicity, including measurements of the effects of lead on enzymes of the hematopoietic system. Lead-induced alterations in blood zinc protoporphyrin (ZPP) and erythrocyte 6-aminolevu-linic acid dehydratase (ALAD) activity have been established as relatively specific biomarkers of lead toxicity to the heme biosynthetic pathway (NRC 1993 USEPA 1986). Increases in blood ZPP occur as a result of inhibition of ferrochelatase (FC) by lead. Inhibition of ALAD by lead, which begins at a blood lead level of about 5 tg/dL (Chisolm et al. 1985 USEPA 1986), is considered to be one of the most sensitive biomarkers currently available. 

Along with blood lead and urine, effects on the heme system can be used as indicators of exposure to lead. Elevations of zinc protoporphyrin (ZPP) or free erythrocyte protoporph)Tin (FEP) can indicate past lead exposures while blood lead will indicate a recent exposure. As shown in Table 22.2, OSFIA requires action when blood lead is 40 pg/100 g of whole blood and removal from lead exposure when the blood lead reaches or exceeds 60 tg/100 g of whole blood or if the average of three consecutive blood lead levels are over 50 pg/lOO g of whole blood. 

Lead also has a toxic effect on the last step in the haem synthesis pathway, which is the insertion of iron into protoporphyrin to create haem. If the enzyme ferrochelatase, responsible for this change, is inhibited by lead, this fails to take place. The amount of protoporphyrin in the erythrocytes, called zinc-protoporphyrin or erythrocyte protoporphyrin (ZZP or EPP), can be used as a measure of this effect. However, it is a less useful measure in studies of the effects of low levels of lead exposure, as an increase in EPP may not be detected at blood levels below about 20jUg/dl (Piomelli et al, 1982), although this threshold for the effect may vary with age (Succop et al,this volume). Other factors, such as iron deficiency, can also cause an increase in EPP levels in the blood... 

Bush, B., Doran, D. and Jackson, K. (1982) Evaluation of erythrocyte protoporphyrin and zinc protoporphyrin as micro screening procedures for lead poisoning detection. Ann. Clin. Biochem., 19, 71-76... 

Zinc-chelated protoporphyrin and free protoporphyrin are extracted from erythrocytes and separated according to their polarity on a reverse-phase system. Their detection by fluorescence is highly specific because both excitation and emission are at a relatively long wavelength. 

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