Barbosa et al. It is well known that Pb affects several enzy- matic processes responsible for heme synthesis. Lead directly inhibits the activity of the cyto- plasmic enzyme δ-aminolevulinic acid dehy- dratase (ALAD), resulting in a negative exponential relationship between ALAD and BPb. Pb depresses coproporphyrinogen oxidase, resulting in increased coproporphyrin activity. Pb also interferes with the normal functioning of the intramitochondrial enzyme ferro- chelatase, which is responsible for the chelation of iron by protoporphyrin. Failure to insert Fe into the protoporphyrin ring results in depressed heme formation and an accumula- tion of protoporphyrin; this in turn chelates zinc in place of Fe, to form zinc proto- porphyrin. These effects also result in modiﬁca- tions of some other metabolite concentrations in urine (ALA-U), blood, (ALA-B) and plasma (ALA-P), coproporphyrin in urine (CP). The activities of pyrimidine nucleotidase (P5´N) and nicotinamide adenine dinucleotide syn- thase (NADS) are also modiﬁed in blood after Pb exposure. Levels of these various metabolites in biological ﬂuids have been used in the past to diagnose Pb poisoning when direct Pb levels were difﬁcult to obtain in tissues or body ﬂuids (Leung et al. 1993) or as information comple- mentary to BPb test results. They are more accurately described as biomarkers for toxic effects of Pb. In this review we focus on mark- ers that are more accurately deﬁned as biomark- ers of Pb exposure, namely, Pb concentrations in biological tissues and ﬂuids. Biomarkers for the toxic effects of Pb have been reviewed in some detail elsewhere (Sakai 2000). Throughout the last five decades, whole blood has been the primary biological fluid used for assessment of Pb exposure, both for screening and diagnostic purposes and for bio- monitoring purposes in the long term. Although BPb measurements reflect recent exposure, they may also represent past expo- sures, as a result of Pb mobilization from bone back into blood (Gulson et al. 1996). In those subjects without excessive exposure to Pb, 45–75% of the Pb in blood may have come from bone (Gulson et al. 1995; Smith et al. 1996). In exposed children, however, it has been reported that the bone-Pb contribution to blood can be 90% or more (Gwiazda et al. 2005). Thus, reductions in BPb levels after environmental Pb remediation may be buffered somewhat by contributions from endogenous Pb sources (Lowry et al. 2004; Rust et al. 1999). Remediation efforts typically result in reductions of BPb levels in exposed children of no more than 30%, when evalu- ated within several months after intervention (U.S. Enviromental Protection Agency 1995). Roberts et al. (2001) reported that in children with BPb levels between 25 and 29 µg/dL who were not treated with chelation drugs, the time required for BPb to decline to < 10 µg/dL is 1670 about 2 years. Some researchers have suggested that the efficacy of Pb hazard remediation efforts should be evaluated over extended peri- ods to allow adequate time for mobilization and depletion of accumulated skeletal Pb stores and to allow a reduction in the absolute contri- bution to BPb levels from these stores (Gwiazda et al. 2005; Lowry et al. 2004). Thus, the mean of serial BPb levels should be a more accurate index of long-term Pb exposure. Data collected as part of the U.S. National Health and Examination Survey (NHANES) give the 95th percentile for BPb as 7.0 µg/dL for children 1−5 years of age and as 5.20 µg/dL for adults 20 years of age and older [U.S. Centers for Disease Control and Prevention (CDC) 2003]. Although the BPb levels of U.S. populations have dropped markedly compared to 30 years ago, new concerns have been raised regarding possible adverse health effects in chil- dren at BPb levels < 10 µg/dL; perhaps there is no safe threshold but, rather, a continuum of toxic effects (Canﬁeld et al. 2003). In light of these concerns, the CDC Advisory Committee on Childhood Lead Poisoning Prevention formed a working group to review the evidence for adverse health effects at BPb levels < 10 µg/dL in children. Although this working group concluded that several studies in the lit- erature had demonstrated a statistically signiﬁ- cant association between BPb levels < 10 µg/dL and some adverse health effects in children, the effects were very small and could conceivably have been inﬂuenced by residual confounding factors. The working group’s report called for further studies to examine the relationship between lower BPb levels and health outcomes to provide a more complete understanding of this issue (CDC 2004). Many studies have reported statistically sig- nificant associations between BPb levels and various health effect outcomes. Some, however, have been statistically weak, with the magni- tude of the effect relatively small. According to Hu et al. (1998), such weaknesses of associa- tion may occur because BPb is not a suffi- ciently sensitive biomarker of exposure or dose at the target organ(s) or because the relation- ships involved are biologically irrelevant and are only found because of an uncontrolled con- founding factor. Furthermore, in view of the kinetics of Pb distribution within the body (cycling among blood, bone, and soft tissues), differentiation of low-level chronic exposure from a short high-level exposure is not possible on the basis of a single BPb measurement (Hu et al. 1998). Consequently, there is renewed interest in alternative biomarkers that may aid diagnosis of the extent of Pb exposure. Such alternatives include Pb determinations in plasma/serum, saliva, bone, teeth, feces, and urine. However, none of these matrices has gained convincing acceptance as an alternative to BPb. This is partly due to data based on erroneous or dubious analytical protocols that do not consider the confounding variables. Plasma/Serum Lead Plasma-Pb likely represents a more relevant index of exposure to, distribution of, and health risks associated with Pb than does BPb. Indeed, from a physiologic point of view, we can assume that the toxic effects of Pb are pri- marily associated with plasma-Pb because this fraction is the most rapidly exchangeable one in the blood compartment. In recent years increased attention has been paid to monitor- ing the concentration of Pb in plasma (or serum). However, research on associations between plasma-Pb and toxicologic outcomes is still sparse, and a signiﬁcant gap in knowl- edge remains. Plasma/serum Pb levels in nonexposed and exposed individuals reported in older publica- tions range widely, from 0.02 to 14.5 µg/L (Versieck and Cornelis 1988). This is proba- bly due to inappropriate collection methods, analytical instrumentation, and methods for Pb determination. The development and use of more sensitive analytical instrumentation, especially inductively coupled plasma mass spectrometry (ICP-MS), has resulted in deter- minations of Pb in plasma and serum speci- mens with much lower detection limits and with better accuracy. More recent data, also based on ICP-MS methods, have shown plasma-Pb levels < 1.0 µg/L in nonexposed individuals (Schutz et al. 1996). The use of advanced analytical techniques is not the only essential requirement to ensure accurate and reliable plasma-Pb data. Contamination of the specimen may occur at the preanalytical phase, namely, during collec- tion, manipulation, or storage. Use of Class-100 biosafety cabinets and clean rooms for specimen preparation and analysis is mandatory. Moreover, all analytical reagents used must be of the highest purity grade. These conditions are far more rigorous than are typically required for clinical BPb measure- ments performed in a commercial laboratory. After the blood specimen has been collected, the serum/plasma separation must be per- formed as soon as possible because there is high potential for Pb to move from the domi- nant BPb subcompartment repository, namely, the erythrocytes, into the plasma via hemolysis, leading to erroneously high results for plasma-Pb. Plasma hemolysis can be esti- mated by analyzing hemoglobin levels in the specimen because these levels are likely to become abnormally elevated with hemolysis (Smith et al. 2002). Materials for specimen collection and storage and the anticoagulant must be of the highest quality because these can be another source of Pb contamination. Commercial evacuated blood tubes, pre- pared speciﬁcally for BPb measurements, are VOLUME 113 | NUMBER 12 | December 2005 • Environmental Health Perspectives