Cumulative Lead Dose and Cognitive Function in Adults: A Review of Studies That Measured Both Blood Lead and Bone Lead

Objective We review empirical evidence for the relations of recent and cumulative lead dose with cognitive function in adults.
In the development of the adult lead management guidelines (see Kosnett et al. 2007), a number of health outcomes adversely affected by lead exposure were discussed. Cognitive function was an important consideration of because of the growing number of studies in this area and increasing concern that cognitive function in adulthood may be affected by relatively low lead doses. In this article, we systematically review recent evidence concerning recent and cumulative lead dose and adult cognitive function.
Measurement of lead dose
In reviewing studies of the health effects of lead, it is critical to understand the available lead biomarkers in terms of how they represent external exposure (in terms of timing, duration, magnitude, and accumulation); how they are influenced by metabolic factors (organ distribution, compartmental dynamics, the influence of physiologic factors); and how the combination of these considerations affects inferences regarding the health effects of lead (Hu et al. 2007). We conclude from these important methodologic issues that the most informative recent epidemiologic studies of lead’s impact on health are those that were able to derive estimates of both recent and cumulative lead exposure for each study participant. To achieve this end with the greatest precision and accuracy, such studies have incorporated measurements of lead in both blood (whole blood, using standard chemical assays such as graphite furnace atomic absorption spectroscopy) and bone [using noninvasive in vivo K-shell X-ray fluorescence (KXRF) instruments].
Blood lead levels measured in epidemiologic studies with valid instruments and standardized calibration and quality control procedures have been reported in the literature for > 35 years. Bone lead levels measured by in vivo KXRF were begun in some research laboratories in the 1980s, but it was not until the mid-1990s that reports began to emerge of KXRF-measured bone lead levels in relation to potential health indicators from epidemiologic studies with sufficient sample sizes (for example, ≥ 100 subjects) to have substantial statistical power. Thus, in this review we summarize all studies to date that measure cognitive function and both blood and bone lead levels (or acceptable surrogate for cumulative lead dose).
Published reviews of relevance to this review
We begin our review with a discussion of three other reviews on the topic of lead dose and cognitive function (Balbus-Kornfeld et al. 1995; Goodman et al. 2002; Meyer-Baron and Seeber 2000). Balbus-Kornfeld et al. (1995) reviewed the evidence on cumulative lead exposure and cognitive function from studies published from 1976 to 1991. Among 21 unique studies that were identified at the time of the authors’ review, none used a biomarker of cumulative dose. Of the four longitudinal studies, all were small (mean sample size in the analysis of 47 lead-exposed subjects), with relatively low follow-up rates and relatively short durations of follow-up. The authors thus concluded that the available literature provided inadequate evidence to conclude whether cumulative exposure or absorption of lead adversely affected cognitive function in adults.
Goodman et al. (2002) and Meyer-Baron and Seeber (2000) are reviewed here because they had generally opposite conclusions, which led to considerable controversy and discussion (Goodman et al. 2001; Schwartz et al. 2002; Seeber and Meyer-Baron 2003; Seeber et al. 2002). The Goodman et al. (2002) article was funded by the German Battery Association, apparently in anticipation of consideration in Germany of lowering the blood lead standard in lead workers (Seeber and Meyer-Baron 2003). Goodman et al. (2002) reviewed 22 studies published between 1974 and 1999 with the expressed aim of evaluating associations between moderate blood lead levels and neurobehavioral test scores after occupational exposure to lead. Studies were included if the central tendency for blood lead levels was < 70 μg/dL, the numbers of exposed and unexposed were reported, and test score arithmetic means and measures of variability were reported for exposed and unexposed workers (Goodman et al. 2002). The authors concluded that none of the individual studies were conclusive or adequate in providing information on the effects of lead on cognitive function and called for prospective studies that would evaluate cognitive function before and after exposure. There was no discussion about whether examining relations of blood lead levels with cognitive function was the most relevant question if the hypothesis was that cumulative lead dose was most important to cognitive function. There was little explicit discussion of whether lead may have acute effects as a function of recent dose, and chronic effects as a function of cumulative dose, or how this could be assessed by review of epidemiologic studies.
Meyer-Baron and Seeber (2000) performed a meta-analysis of 12 studies using selection criteria similar to Goodman et al. (2002) but also with the requirement for reporting means and standard deviations of dependent variables (Meyer-Baron and Seeber 2000). They concluded that there were obvious neurobehavioral deficits at current blood lead levels < 40 μg/dL. Again, the focus was on associations with blood lead levels, and there was little formal discussion about which lead biomarker was most relevant to hypotheses about how cumulative lead dose may influence cognitive function. Thus, this is the first review to evaluate epidemiologic studies that distinguish between the acute effects of recent dose from the chronic effects of cumulative dose.
Methods
Methodologic considerations for relations of lead dose and cognitive function
Many methodologic issues of relevance to the epidemiologic investigation of lead and cognitive function have been addressed elsewhere in this minimonograph (Hu et al. 2007). When evaluating the associations of cumulative lead dose with cognitive function, it is important to acknowledge that nonoccupational sources of lead exposure were present for all members of the general population, including lead workers throughout the early part of this century until public health interventions progressively removed lead from gasoline and many consumer products during the 1970s and 1980s (Agency for Toxic Substances and Disease Registry 1999; Annest et al. 1983; Pirkle et al. 1998). Lead remains a low-level and ubiquitous neurotoxicant in the environment and is found in measurable levels in all individuals (Hoppin et al. 1995). Thus, current tibia lead levels represent a mix of occupational and environmental exposures. This review does not try to determine whether the main source of lead was occupational or environmental but rather focuses on whether lead in blood or bone is associated with adverse cognitive outcomes in adults.
Identification of studies
We conducted a systematic literature review of the association between blood and bone lead biomarkers and cognitive functioning in adults. Our aim was to select studies that compared markers of both recent and cumulative lead dose in their relations with cognitive function. Both occupationally and environmentally exposed adult populations were included. We searched the PubMed (National Library of Medicine 2006) and PsycINFO databases (American Psychological Association 2006) for epidemiologic studies using keywords such as blood, bone, lead, cumulative, cognitive, and neurobehavior. There were no date or language restrictions. From the identified publications and relevant review articles, we examined reference lists to locate additional studies that measured both recent and cumulative lead dose. This includes blood lead levels, bone lead levels, or a surrogate measure of cumulative lead dose such as integrated blood lead (IBL), area under the curve of blood lead levels over time, or the product of blood lead level and employment time. Studies were not considered for the review if they a) contained no original research, b) were conducted on nonhuman subjects, c) were case reports, d) contained no standardized neurocognitive assessment outcomes, or e) lacked measures of both recent and cumulative lead dose.
Data abstraction
We abstracted data from articles meeting the selection criteria. Study quality was assessed with the following criteria: a) exposure was assessed at an individual level; b) exposure was assessed with a biomarker; c) cognitive outcomes were objective, standardized tests; d) statistical adjustment for potential confounders including age, sex (in studies with both men and women), and education; e) data collection was similar in exposed and nonexposed participants; f ) time period of study was the same in exposed and nonexposed participants; and g) there was a detailed description of the approach to data analysis. We decided not to try to derive a pooled estimate across studies of the associations of lead dose biomarkers with cognitive function because of differences in methods for subject selection, blood and bone lead measurements, neurobehavioral outcomes, approach to regression modeling, and presentation of results across studies. Pooled estimates from metaanalysis also can be highly influenced by decisions regarding how and whether to pool certain results. We thus decided to present details for each study and discuss them in turn.
Results
Overview of evidence
We identified three main types of studies that reported cross-sectional or longitudinal associations of blood and bone lead levels with cognitive function. These were of a) environmentally exposed individuals in the general population, b) workers with current occupational exposure, and c) former lead workers without current occupational exposure to lead. We have summarized these studies in Table 1, provided details in Table 2, and discuss them in order below.
Studies of adults without occupational lead exposure
We identified six articles from three studies [i.e., residents near a lead smelter, the Normative Aging Study (NAS), and the Baltimore Memory Study] that evaluated subjects with mainly environmental exposure to lead (Tables 1 and 2). One study of young adults 19–29 years of age compared 257 individuals with high childhood blood lead levels from exposure 20 years previously from a lead smelter to 276 age- and sex-matched controls. This study found impairment on many cognitive tests among the highly exposed group, but minimal association on most tests with tibia lead levels measured during young adulthood (Stokes et al. 1998).
Four articles from the NAS reported associations of blood and bone lead levels in a cohort of older men. One of these articles (Payton et al. 1998) was a first report that examined scores on a large battery of cognitive tests of a small sample (n = 141) of NAS participants. This was subsequently followed up with a report on a much larger number of NAS participants (n = 1,089 with blood lead levels and n = 760 with bone lead levels, 412–515 of whom took different tests twice approximately 3.5 years apart) (Weisskopf et al. 2007). Cross-sectional analyses in the original report found that increased blood lead levels across a relatively low range of levels [mean ± SD = 5.5 ± 3.5 μg/dL) were a stronger predictor, compared with tibia or patella lead levels, of poorer performance on tests of speed, verbal memory, vocabulary, and spatial copying skills. However, this was not confirmed in the larger, cross-sectional analysis, except possibly for scores on a vocabulary test (Weisskopf et al. 2007). Conversely, in longitudinal analyses, the larger study found more decline over time on almost all cognitive tests associated with both higher patella and higher tibia bone lead levels, with the associations reaching statistical significance for pattern comparison and spatial copying skills. An earlier, similar longitudinal analysis by Weisskopf et al. (2004) in this same population reported that patella lead levels were significantly associated with a decline in Mini-Mental State Examination (MMSE; Folstein et al. 1975) score over time. A slightly smaller association was observed with tibia lead levels, whereas no association was observed with blood lead levels. In cross-sectional analyses of the same population, higher blood lead levels were a stronger predictor of poorer performance on the MMSE, as were higher patella and tibia bone lead levels (Payton et al. 1998; Wright et al. 2003).
In a study of almost 1,000 persons 50–70 years of age randomly selected from the general population in the Baltimore Memory Study (BMS), a cross-sectional analysis showed that relatively low current blood lead levels were not associated with cognitive domain scores. However, moderate tibia lead levels (mean ~ 19 μg/g) were significantly associated with worse performance in all seven cognitive domains (Shih et al. 2006). Thus, in the environmental studies of older adults, the most consistent findings across studies are associations between bone lead levels and cognitive function. The associations in the BMS were cross-sectional, whereas the predominant associations in the NAS were with change in cognitive function over time, although a significant cross-sectional association with MMSE score was also observed in this sample. Taken together, these data suggest that at environmental exposure levels, the effects of cumulative exposure are more pronounced than recent effects of current exposure. The absence of associations in the Stokes et al. (1998) study could be because of the younger age of studied subjects, the very low current blood and tibia lead levels, or the inadequacy of tibia lead in the third decade of life to estimate early life dose (Hoppin et al. 2000).
Studies of occupationally exposed workers
Fifteen articles were identified of workers with current or past occupational exposure to lead. Eight of these studies used a surrogate measure of cumulative lead dose (i.e., IBL) rather than a direct measure of lead in bone. Among these studies, which compared blood and IBL lead dose, when the lead exposure was primarily current (e.g., relatively high blood lead levels), most studies found an association between increasing blood lead values and worse cognitive function (Barth et al. 2002; Bleecker et al. 1997; Lucchini et al. 2000). However, studies in which the exposure was primarily in the past demonstrated that surrogate measures of cumulative dose were a stronger predictor of worse cognitive function compared with blood lead levels (Bleecker et al. 2005; Chia et al. 1997; Lindgren et al. 1996). Studies that used bone lead levels as a direct indicator of retained cumulative lead dose are summarized below.
One study of currently exposed lead workers in South Korea (n = 803) found strong and consistent associations of blood lead levels with worse cognitive function after adjustment for covariates, but tibia lead levels were not as consistently associated (Schwartz et al. 2001). The same null findings for bone lead levels were observed in two smaller studies, one with male smelter workers (n = 57) in whom finger bone (mixed trabecular and cortical tissue) lead levels were measured (Osterberg et al. 1997). The second article describes the study of a sample of 54 storage battery workers in whom tibia and calcaneus lead levels were measured (Hanninen et al. 1998). This is the only study published to date to report an association between IBL and cognitive outcomes in which there was a lack of an association with bone lead levels. Both these studies used early XRF techniques (e.g., KXRF with cobalt-57) with higher limits of detection that have not been commonly used since, and this use makes the findings more difficult to interpret. Bleecker et al. (1997), in a study similar to the one by Schwartz et al. (2001), reported stronger and more consistent associations of blood lead measures and neurobehavioral test performance compared to tibia lead levels.
In the South Korean lead workers with current occupational exposure, a longitudinal analysis was performed to separate recent lead dose (measured as blood lead levels) from cumulative lead dose (measured as tibia lead levels), and acute effects from chronic effects in 575 subjects with complete data across the three study visits (Schwartz et al. 2005). The authors reported significant cross-sectional associations of blood lead levels with lower executive ability and manual dexterity test scores, with some evidence also for a longitudinal association of changes in blood lead levels with neurobehavioral decline. Tibia lead levels were more consistently associated with longitudinal declines in manual dexterity, executive abilities, neuropsychiatric symptoms, and peripheral sensory functioning than change in blood lead levels. The authors concluded that lead was associated with worse cognitive function in two ways: an acute effect of recent dose and a chronic effect of cumulative dose. The authors also discussed that contrasting associations with blood and tibia lead levels could be due to the following: a) tibia and blood lead levels are biologically related and blood lead is in equilibrium with bone lead stores; b) the error in measurement of tibia lead levels is larger than that for blood lead; c) controlling for cross-sectional associations could obscure longitudinal ones; and d) lead in blood reflects recent external exposure, and is in equilibrium with bone lead stores, possibly taking away explained variance from bone lead associations via this correlation in cross-sectional analyses.
Results of a cross-sectional analysis of former organolead workers showed that higher peak tibia lead levels (range, –2.2 to 105.9 μg/g) were related to poorer functioning on a number of cognitive tests, including those assessing manual dexterity, executive ability, verbal intelligence, and verbal memory (Stewart et al. 1999). In a longitudinal analysis in this same population, among 535 lead workers exposed a mean of 16 years before, increases in peak tibia lead levels [mean ± SD = 22.6 ± 16.5 μg/g] but not in blood lead levels predicted declines over time in these same domains in addition to visual memory (Schwartz et al. 2000). This finding indicates that even many years after high lead exposure, and in the absence of high current lead exposure, cumulative lead dose may exert progressive effects on cognitive functioning (Links et al. 2001).
Lead exposure and psychiatric symptoms
Several lines of evidence suggest that increased blood lead levels are associated with psychiatric symptoms in adults, such as depression, anxiety, irritability, and anger. For example, a cross-sectional analysis of 107 occupationally exposed individuals showed increased rates of depression, confusion, anger, fatigue, and tension as measured by the Profile of Mood States (POMS; McNair et al. 1971) among those with blood levels > 40 μg/dL (Baker et al. 1983). Maizlish et al. (1995) found that current and cumulative measures of blood lead levels in currently exposed lead workers were associated with tension, anxiety, hostility, and depression measured by the POMS questionnaire. Lindgren et al. (1996) examined the POMS’ factor structure in retired lead smelter workers and showed that the resulting “general distress” factor was significantly related to IBL but not to current blood lead level.
In occupationally exposed South Korean lead workers, tibia lead levels were significantly associated with more depressive symptoms measured by the Center for Epidemiologic Studies Depression scale (CES-D; Radloff 1977) after adjusting for age, sex, education, job duration, and blood lead level (Schwartz et al. 2001). However, only one recent study has examined a direct measure of cumulative dose with bone measurements in a community sample (Rhodes et al. 2003). These authors used the Brief Symptom Inventory (BSI; Derogatis and Melisaratos 1983) to show that patella bone lead levels were associated with an increased risk of anxiety and depression sub-scale scores. The logistic regression estimate for the phobic anxiety subscale was statistically significant (p < 0.05), as well as for the combined measure of all three BSI subscales (anxiety, depression, and phobic anxiety).
Psychiatric symptoms, specifically symptoms of depression, potentially share the same neural substrates with components of cognition, and thus may be important to late-life cognitive functioning. Compared with nondepressed elderly individuals, depressed elderly perform more poorly on tests involving attention, memory encoding, and retrieval. However, intelligence tests are more resistant to these effects of depression (Arnett et al. 1999; Naismith et al. 2003; Weingartner et al. 1981). Depressive symptoms (as measured by the CES-D) are positively associated with both the risk of Alzheimer disease and a steeper rate of cognitive decline (Wilson et al. 2002). Because late-life symptoms of depression are closely associated with dementia, investigators have put forth a number of hypotheses that suggest depression a) may be a risk factor for cognitive decline, b) has risk factors in common with dementia, c) is an early reaction to declining cognition, and d) influences the threshold at which dementia emerges [for review see Jorm (2000)]. The exact temporal and mechanistic relation remains unclear. Regardless of the exact relation between depressive symptoms and cognitive function, however, the assessment of the impact of lead exposure on these outcomes is not compromised. Whatever the associations with these outcomes, they would still be attributed to lead—that is, even if depressive symptoms lead to worse cognitive performance, and lead leads to symptoms of depression, the cognitive impairment as a result of that depression could still be considered part of the total effect of lead.
Lead–gene interactions
In the former organolead worker studies discussed above, possessing at least one apolipoprotein E (APOE) ɛ 4 allele magnified the negative cross-sectional association of tibia lead levels with performance on the cognitive domains of executive ability, manual dexterity, and psychomotor skills (Stewart et al. 2002). No direct effects of the APOE ɛ 4 allele were observed on cognitive function in this study, presumably because of the sample’s younger age (range, 41–73 years). Other studies have found that APOE ɛ 4 modifies dementia outcome in individuals with previous traumatic head injury, suggesting that APOE ɛ 4 plays a role in recovery from brain insults (Mayeux et al. 1995), which may be extended to include insult from lead exposure.
Discussion
Summary of evidence for a causal relationship
The literature on associations of recent and cumulative dose biomarkers with cognitive function has grown impressively since the 1995 review (Balbus-Kornfeld et al. 1995). We believe sufficient evidence exists to conclude that there is an association between lead dose and decrements in cognitive function in adults. Overall, while the association between blood lead levels and cognitive function is more pronounced in occupational groups with high current lead exposures, associations between bone lead levels and cognitive function are more evident in studies of older subjects with lower current blood lead levels, particularly in longitudinal studies of cognitive decline.
Consistency of associations
Following is a summary of the findings from each of the three types of populations. First, cross-sectional studies of currently exposed lead workers showed that associations of blood lead levels and cognitive function were clearer than the associations for tibia, patella, or calcaneus lead levels, perhaps because the acute effects of recent dose in an occupational setting masked the chronic effects of cumulative lead dose. Second, previously exposed occupational populations demonstrated a stronger association between cumulative lead dose measured in tibia bone with cognitive deficits compared with blood lead levels. The two studies that deviated from these otherwise consistent findings may not have had sufficient power to detect any associations (n < 60). Last, studies of environmentally exposed adults who had notably higher exposures in the past suggest that bone lead level is more consistently associated with performance on cognitive tests than is blood lead level. The domains associated with lead dose do not differ in general by lead biomarker (blood, tibia, patella). The cognitive domains consistently associated with each biomarker in both environmental and occupational studies on adults include verbal and visual memory, visuospatial ability, motor and psychomotor speed, manual dexterity, attention, executive functioning, and peripheral motor strength. Comparisons of lead and psychiatric symptom associations in previously and currently exposed samples lend credence, although perhaps at higher thresholds than for cognitive outcomes, that neurobehavioral functioning is consistently associated with blood lead when exposure is currently high (e.g., occupational) and bone lead when exposure is primarily from past chronic exposure.
These associations exist in multiple settings, including both occupational and non-occupational, in men and women, and in populations with diversity by socioeconomic status and race/ethnicity. This reduces the likelihood of associations by statistical chance or due to unmeasured confounding. However, this consistency cannot completely rule out the possibility of uncontrolled confounding or effect modification (Martin et al. 2006; Shih et al. 2006). In addition, in studies of general populations with diversity by socioeconomic status and race/ethnicity, the ability to disentangle social, cultural, and biological factors from the “independent” influence of lead dose may be a futile exercise (Weiss and Bellinger 2006).
Strength of association
The strength of associations between lead and cognitive function is strong and can be compared to the influence of age on cognitive function. The comparative magnitude of these effects has been reported in several studies. In currently exposed lead workers, cross-sectional associations showed that a 5-μg/dL increase in blood lead was equivalent to an increase of 1.05 years in age (Schwartz et al. 2001). The magnitude of cross-sectional associations with tibia lead levels in the BMS was moderate to large. A proportion comparison of the direct effect of age and the direct effect of tibia lead levels on cognitive outcomes demonstrated that the magnitude of the association with tibia lead levels was moderate to large, equivalent to 22–60% of the magnitude of the age effect in its relations with cognitive domain scores. Specifically, an interquartile range increase in tibia lead levels was equivalent to 2–6 more years of age at baseline across all seven domains (Shih et al. 2006).
Longitudinal analyses in the NAS observed that an interquartile range higher patella lead level was approximately equivalent to that of aging 5 years in relation to the baseline MMSE score (Weisskopf et al. 2004) and an interquartile range higher bone (patella or tibia, depending on the specific cognitive outcome) lead level was approximately equivalent to that of aging 1 year in relation to the baseline test scores on a battery of cognitive tests (Weisskopf et al. 2007).
Specificity
Lead has adverse effects on many other health outcomes in addition to cognitive function. This is not surprising given lead’s numerous biologic effects, including calcium agnonism and antagonism (Ferguson et al. 2000), binding to sulfhydryl and carboxyl groups on proteins, and activation of nuclear transcription factors (Ramesh et al. 2001), for example. It is thus not surprising that lead’s toxicity is not specific to the brain and we do not believe this lack of target organ specificity diminishes the inference for a causal relationship between lead and cognitive dysfunction.
Temporal relationship
Associations between lead biomarkers and cognitive outcomes have been demonstrated in both cross-sectional and longitudinal studies. In several of the longitudinal studies, change in cognitive function was explicitly modeled in relation to preceding lead dose or in relation to change in lead dose. In either case, the temporality condition is met. In addition, as bone lead is a measure that ascertains prior dose, even in cross-sectional analyses, analysis of bone lead with cognitive test scores evaluates lead dose that preceded current cognitive performance; thus, while cognitive assessment is cross-sectional, dose assessment is retrospective and cumulative. This again would minimize concerns about incorrect temporal relations.
Biological gradient (dose–effect relations)
Nearly all reviewed studies found a dose–effect relation for blood lead, bone lead, or both. Existing studies do not allow determination of a threshold dose for either blood lead or bone lead or the shape of the dose–effect relationship at low dose levels. Associations have been observed in populations with mean blood lead levels as low as 4.5 μg/dL (Wright et al. 2003) and mean tibia lead levels as low as 18.7 μg/g (Shih et al. 2006).
Biologic plausibility and experimental data
Lead adversely affects the brain in a variety of ways. Lead is thought to increase oxidative stress, induce neural apoptosis, influence neurotransmitter storage and release, and damage mitochondria. The ability of lead to substitute for calcium allows it to affect calcium-mediated processes and pass through the blood–brain barrier. It may also interfere with zinc-dependent transcription factors, altering the regulation of genetic transcription (Zawia et al. 2000). Animal studies indicate that the accumulation of lead in the brain is generally uniform (Widzowski and Cory-Slechta 1994), although the hippocampus and limbic system, prefrontal cerebral cortex, and cerebellum are clearly principal sites of the effects of lead (Finkelstein et al. 1998). Low lead levels in rats produce structural changes in the hippocampus (Cory-Slechta 1995), a brain region critical for learning and memory (Eichenbaum 2001), which is consistent with the finding of learning and memory deficits in lead-exposed individuals.
Blood lead level is a measure of current biologically active lead burden and is therefore a better marker of the acute effects of recent lead dose. These are likely to be effects on neurotransmission and calcium enzyme-dependent processes such as synaptic plasticity. This could lead to circulating blood lead impairing, for example, information storage and retrieval mechanisms or processing speed, which have been suggested to impair performance on cognitive tests (Salthouse 1996a, 1996b). Lead levels in bone are a measure of cumulative dose over decades as well as a source of lead in the body that is available for mobilization into blood, especially during periods of increased bone turnover (e.g., pregnancy, puberty). Although lead stored in bone is not directly harmful to the brain, the cumulative effects of chronic lead exposure are likely to be related to oxidative stress and neuronal death and could impair cognitive function, for example, by reducing the capacity of specific regions to process information, or by impairing diffuse ascending projection systems such as the midbrain cholinergic and dopaminergic cells.
Lead may also influence cognitive function indirectly through its effects on blood pressure, hypertension, or homocysteine levels. Increased homocysteine levels, a well-known risk factor for cardiovascular disease, have also been associated with risk for poorer cognitive functioning (Dufouil et al. 2003; Schafer et al. 2005a) and risk for dementia (Hogervorst et al. 2002; McCaddon et al. 2003; Selley 2003). Homocysteine is neurotoxic to the central nervous system by influencing neurotransmitter synthesis, and causing excitotoxicity and cell death (McCaddon and Kelly 1992; Parnetti et al. 1997). Blood lead levels were associated with homocysteine levels as well, although the direction of causality has yet to be determined (Guallar et al. 2006; Schafer et al. 2005b). Both blood and bone lead levels have been linked with blood pressure and hypertension in community-based samples of older adults (Martin et al. 2006; Nash et al. 2003) and occupationally exposed populations (Glenn et al. 2003, 2006). Hypertension has also been identified as a potential risk factor for dementia (Birkenhager and Staessen 2006; Hayden et al. 2006; Skoog and Gustafson 2006). Thus, lead may indirectly play a role in cognitive declines by way of poor vascular health.
We believe the effect modification by APOE genotype offers strong biologic plausibility to the inference that lead causes cognitive dysfunction (Stewart et al. 2002). The APOE ɛ 4 allele is a risk factor for lateonset Alzheimier disease (Corder et al. 1993; Meyer et al. 1998; Saunders et al. 1993), hippocampal atrophy (Moffat et al. 2000), and senile plaques (Zubenko et al. 1994). It appears that the APOE ɛ 4 allele lowers the age of onset of the disease and accelerates age-related cognitive decline (Meyer et al. 1998). Mechanistically, APOE ɛ 4 is involved in the recovery response of injured nerve tissue (Poirier and Sevigny 1998), with the APOE ɛ 4 allele having reduced ability to promote growth and reduced antioxidant properties (Miyata and Smith 1996; Teter et al. 1999; Yankner 1996). The interaction of APOE genotype with tibia lead level may be related to an impaired ability to counteract injury from lead exposure among APOE ɛ 4 carriers.
Another recent study also offers biologic plausibility. In the former organolead workers, tibia lead level was associated with the prevalence and severity of white matter lesions on brain MRI, using the Cardiovascular Health Study white matter grading system (Stewart et al. 2006). Tibia lead level was also associated with smaller volumes on several regions of interest ranging from large (e.g., total brain volume, lobar gray and white matter volumes) to small (e.g., cingulate gyrus, insula, corpus callosum). As volume can decline because of changes in cell number, synaptic number or density, or other changes in cellular architecture, these findings reinforce evidence that lead may cause a persistent change in the brain that is associated with progressive declines in cognitive function.
Public health implications
The removal of lead from gasoline, paint, and most other commercial products has succeeded in dramatically reducing environmental sources of lead exposure, and this has been reflected by the parallel declines in mean blood lead levels in Americans over the same time frame. However, lead has accumulated in the bones of older individuals, and especially those of lead workers exposed at the continued higher levels encountered in lead-using workplaces. Thus, past use of lead will continue to cause adverse health effects even when current exposures to lead are much lower than in the past. Lead in bone is not directly harmful to the central nervous system, and most of the structural and neurochemical damage is likely to have occurred decades ago. Nevertheless, lead in bone might serve as a source from which lead can be mobilized into blood, and potentially cross the blood–brain barrier. The chronic effects of lead may account for a proportion of cognitive aging; future research will be able to determine whether the chronic effects of cumulative lead dose alter the trajectory of normal cognitive aging. Research efforts should be directed to development of preventive interventions for both lead-associated cognitive decline with aging from past exposures, as well as the mobilization of current bone lead stores into the circulatory system leading to new health effects.
Cognitive aging occurs in conjunction with the normal biological aging process. It remains to be determined whether lead affects cognitive aging in adults by permanently reducing brain circuitry capacity thereby lowering baseline cognitive functioning, or by inducing steeper declines in cognitive functioning, leading to abnormal cognitive aging. It may be that lead influences cognitive health through its relationship with depressive symptoms, hypertension, or homocysteine levels, all of which influence cognitive impairment and risk of dementia. Future investigations should explicitly account for these complex causal pathways, and also determine whether chronic effects of cumulative lead dose increases the risk for such clinically relevant syndromes as mild cognitive impairment (Petersen et al. 1999).

lead
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neurobehavior


ADAMTS13 phenotype in plasma from normal individuals and patients with thrombotic thrombocytopenic purpura

The activity of ADAMTS13, the von Willebrand factor cleaving protease, is deficient in patients with thrombotic thrombocytopenic purpura (TTP). In the present study, the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting. Normal plasma (n = 20) revealed a single band at 190 kD under reducing conditions using a polyclonal antibody, and a single band at 150 kD under non-reducing conditions using a monoclonal antibody. ADAMTS13 was not detected in the plasma from patients with congenital TTP (n = 5) by either antibody, whereas patients with acquired TTP (n = 2) presented the normal phenotype. Following immunoadsorption of immunoglobulins, the ADAMTS13 band was removed from the plasma of the patients with acquired TTP, but not from that of normal individuals. This indicates that ADAMTS13 is complexed with immunoglobulin in these patients. The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis, impaired cellular secretion, or enhanced degradation in the circulation. This study differentiated between normal and TTP plasma, as well as between congenital and acquired TTP. This method may, therefore, be used as a complement in the diagnosis of TTP.
Introduction
Von Willebrand factor (VWF) is a glycoprotein that plays a key role in the primary hemostatic process by inducing platelet adhesion and aggregation at sites of vascular injury under conditions of high shear stress. The main source of circulating VWF is the endothelium, from which it is secreted in the form of ultra-large multimers (ULVWF) [53]. ULVWF multimers are biologically very active [2, 31] and, upon release, undergo processing into smaller multimers in normal individuals. This occurs on the surface of endothelial cells [10]. VWF defects may potentially lead to both bleeding and thrombotic disorders: defective VWF secretion, intravascular clearance, multimer assembly, or increased proteolytic degradation may lead to different types of von Willebrand disease. On the other hand, dysfunctional VWF proteolysis may lead to the thrombotic disorder thrombotic thrombocytopenic purpura (TTP) [40].
TTP is a thrombotic microangiopathy (TMA) characterized by microangiopathic hemolytic anemia, thrombocytopenia, fever, neurological and renal manifestations. Chronic recurrent TTP has been associated with the presence of ULVWF in the plasma [30]. ULVWF multimers are capable of inducing increased platelet retention in children with TTP [21]. These observations, along with the finding of VWF and platelet-rich (but fibrin-poor) thrombi in the microcirculation of the heart, brain, kidneys, liver, spleen, and adrenals in TTP patients [3], led to the conclusion that ULVWF multimers are responsible for the disseminated platelet thrombi occurring in TTP and that their degradation to smaller VWF multimers is impaired due to the deficiency of a VWF-cleaving protease [15].
Recently, the VWF-cleaving protease was purified [12, 13, 16, 50] and the encoding gene sequenced, linking the protease to the ADAMTS (a disintegrin-like and metalloprotease with thrombospondin-type-1 motif) family of metalloproteases [27]. The protease, named ADAMTS13, cleaves VWF at the 1605Tyr-1606Met peptide bond in the A2 domain, yielding the 140-kD and 176-kD VWF fragments present in normal plasma [13, 50]. Cleavage is made possible by a conformational change in VWF due to shear stress in the circulation, which exposes the cleavage site, making it susceptible to proteolysis [55]. ADAMTS13 activity is severely deficient (<5% of normal plasma activity) in TTP patients [6], either due to a mutation in the ADAMTS13 gene in the congenital form of TTP or due to auto-antibodies in the acquired form [14, 27, 52]. Autosomal recessive hereditary TTP (also termed the Upshaw-Schulman syndrome) typically presents during the neonatal period or early childhood (<10 years of age), but may also manifest during adolescence and adulthood. Recurrent TTP episodes may occur as often as every third week. TTP recurrences are associated with cerebral vascular accidents in approximately 30% of cases, and these episodes may lead to neurological complications. Renal manifestations may be mild or may result in acute renal failure due to hemoglobinuria and TMA. About 20% of patients progress to end-stage renal failure [28].
Hemolytic uremic syndrome (HUS) is a similar microangiopathic disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure [5]. Two forms of HUS have been described: D+ or typical (diarrhea-associated) HUS and D- or atypical (non-diarrhea-associated) HUS. D+ HUS occurs after infection with Shiga-like toxin producing bacteria, typically, enterohemorrhagic Escherichia coli. The patients are usually children presenting with abrupt onset of diarrhea, followed by the development of HUS 2–10 days later. A prothrombotic state precedes the acute renal failure [8], but the pathogenetic mechanism is, as yet, unclear. It is assumed that bacterial virulence factors gain access to the circulation, circulate on blood cells, activate platelets, and reach the kidney, where the endothelium is injured [36, 47]. D- HUS is associated with mutations in certain complement regulatory factors, such as factor H, factor I, and membrane co-factor protein (CD46). The mutations lead to activation of the complement system on host endothelial cells [29, 58]. The resulting vascular damage may lead to the formation of thrombotic lesions in the kidneys.
Although HUS patients are, typically, young children with a history of diarrhea and acute renal failure, the clinical manifestations of HUS and TTP often overlap, making differentiation between the two syndromes based solely on clinical presentation difficult. ADAMTS13 antigen levels can differentiate between HUS and TTP, as they are severely deficient in patients with congenital TTP and normal to moderately reduced in HUS [51]. Assays for ADAMTS13 activity can, therefore, differentiate between TTP (congenital and acquired) and HUS [26, 48].
Several ADAMTS13 assays are available today based on antigen detection and activity [15, 17, 25, 38], showing the presence of the protease (by enzyme-linked immunosorbent assay, ELISA) and its bioactivity in normal plasma and the lack of protease and activity in the plasma from patients with congenital TTP. These assays have also shown that patients with acquired TTP have auto-antibodies that neutralize the activity of ADAMTS13. The present study utilized a different method, immunoblotting, and two anti-ADAMTS13 antibodies against specific domains, to investigate the presence of ADAMTS13 antigen in normal plasma, TTP plasma (congenital and acquired), and in heterozygous carriers of ADAMTS13 mutations, demonstrating the presence of ADAMTS13 and its size in normal plasma, the lack thereof in congenital TTP, and auto-antibody-bound protease in acquired TTP.
Materials and methods
Subjects
Citrated plasma was available from patients with congenital (n = 5) and acquired (n = 2) TTP. The patient data are presented in Table 1. The ADAMTS13 activity level was assayed as previously described [15, 17].
Table 1Clinical and laboratory data regarding thrombotic thrombocytopenic purpura (TTP) patientsPatient no.SexAge at debut Age at sampling (years)Current age (years)Symptoms during episodesNo. of episodesADAMTS13 activity levelADAMTS13 mutationADAMTS13 inhibitor Reference1aM2 d1619Jaundice, hemolytic anemia, thrombocytopenia, macroscopic hematuria, pathological urinalysis, fever, neurological symptoms, elevated serum creatinine>5<5%4143insAbcNone [4, 20, 43]2aM5.3 y1718Jaundice, hemolytic anemia, thrombocytopenia, fever, neurological symptoms, pathological urinalysis>5<5%4143insAbcNone[4, 20, 43]3F20 m1523Hemolytic anemia, thrombocytopenia, hematuria, epileptic attacks, slightly elevated serum creatinine>5<5%P353Ld, P457LeNone[4]4M3 y79Hemolytic anemia, thrombocytopenia, purpura, pathological urinalysis4<5%P671Lf, 4143insANone[43]5M2 d3939Jaundice, hemolytic anemia, petechiae, thrombocytopenia, transitory neurological deficits and aphasia, elevated creatinine>5<5%4143insAbNone–6F54 y7075Recurrent hemolytic anemia and thrombocytopenia, reduced consciousness, pathological urinalysis>5<5%NA0.5 U/ml–7F25 y4244Thrombocytopenia, hemolytic anemia, elevated creatinine during viral infection and pregnancy2<5%NA0.2 U/ml–aPatients 1 and 2 are siblingsbPatients 1, 2, and 5 are homozygous for the ADAMTS13 mutationc4143insA leads to a mutation in the second CUB domaindP353L is a mutation in the disintegrin-like domaineP457L is a mutation in the cysteine-rich domainfP671L is a mutation in the spacer domainNA: not assayed
The study also included the parents of patients 1–4. The parents of patients 1 and 2 are both heterozygous for the 4143insA mutation, and have protease activity levels of 20% (mother) and 50% (father), as assayed by the VWF multimeric structure analysis [15]. The parents of patient 3 are heterozygous for the P353L (mother) and P457L (father) mutations, and both have 50% ADAMTS13 activity. The parents of patient 4 are heterozygous for P671L (mother) and 4143insA (father), and have 50% ADAMTS13 activity. All parents are clinically unaffected.
Plasma samples from 20 healthy adult volunteers were used as controls. The study was conducted with the approval of the ethics committee of Lund University and the plasma samples were collected with the informed consent of the patients, their parents, and the controls.
Plasma samples
At the time of sampling, the patients were treated regularly with fresh frozen plasma or Octaplas (Octapharma, Stockholm, Sweden; patients 1–5). Patient 6 was treated with plasma infusions every sixth week. Patient 7 did not receive any plasma treatment at sampling. All blood samples were obtained at least three weeks (patients 1–4 and 6) or one week (patient 5) after the last treatment. Venous blood from patients and controls was collected, and the plasma obtained as previously described [20].
Anti-ADAMTS13 antibodies
A polyclonal anti-peptide antibody was raised in New Zealand white rabbits against a unique sequence in the second CUB domain (AA1413-1427) and affinity-purified against the peptide. Antibody specificity was tested by ELISA (plates coated with the peptide) and by immunoblotting with purified plasma ADAMTS13 [50] under reducing (SDS-PAGE) and non-reducing (dot blot) conditions.
The monoclonal antibody A10 [56], directed against the disintegrin-like domain, was used to confirm the results of the polyclonal antibody. The polyclonal antibody reacted with ADAMTS13 under reducing (reduced by the addition of 2-mercaptoethanol to disrupt disulfide bonds in the protease) and non-reducing conditions, whereas the monoclonal antibody reacted with ADAMTS13 only under non-reducing conditions.
Immunoblot analysis for the detection of ADAMTS13 in plasma
The plasma samples (1:20) were subject to SDS-PAGE under reducing (for blotting with the polyclonal antibody) and non-reducing conditions (for blotting with the monoclonal antibody) [19, 58]. Purified plasma ADAMTS13 (1:100) was used as the control for the polyclonal antibody. Immunoblotting was performed with rabbit anti-ADAMTS13 IgG 1.6 μg/ml followed by goat anti-rabbit IgG HRP (DakoCytomation, Carpinteria, CA) 1:2000, or with mouse anti-ADAMTS13 IgG 0.6 μg/ml followed by goat anti-mouse IgG HRP (DakoCytomation, Carpinteria, CA) 1:2000. The signal was detected by chemiluminescence. The specificity of the signal obtained with the polyclonal antibody was tested by preincubation with a 50-fold molar surplus of blocking peptide followed by immunoblotting with the blocked antibody. The specificity of the secondary antibodies was tested by omission of the primary antibodies.
Immunoblotting with the polyclonal antibody revealed, in addition to ADAMTS13, two unspecific bands at 130 kD and 170 kD, which were identified as C3 and alpha-2-macroglobulin. These proteins were removed by incubating the plasma samples with protein A-sepharose coupled rabbit anti-C3 IgG and rabbit anti-alpha-2-macroglobulin IgG. The results obtained using the polyclonal antibody show samples from which these proteins have been removed.
In order to investigate the presence of ADAMTS13-autoantibody complexes in the plasma from patients with acquired TTP, samples were passed onto protein G-sepharose (Amersham Biosciences, Buckinghamshire, UK) prior to immunoblotting. Normal plasma (n = 2) was used for comparison.
Results
Detection of ADAMTS13 in plasma samples from normal individuals
Normal plasma under reducing conditions revealed a single immunoreactive band at 190 kD when blotted against the polyclonal antibody (Fig. 1a). Purified plasma ADAMTS13 showed a similar band under the same conditions (Fig. 1a). The monoclonal antibody detected an immunoreactive band at 150 kD in normal plasma under non-reducing conditions (Fig. 1b). Preincubation of the polyclonal antibody with the blocking peptide abolished the ADAMTS13 band in normal plasma (data not shown). Immunoblots in which the primary antibodies had been omitted showed no bands (data not shown).
Fig. 1a–gDetection of ADAMTS13 in normal and thrombotic thrombocytopenic purpura (TTP) plasma. a The polyclonal antibody detected a single band at 190 kD in normal plasma (NP) under reducing conditions. Purified plasma ADAMTS13 in the right lane showed a similar band. b Normal plasma under non-reducing conditions revealed an immunoreactive band at 150 kD when blotted against the monoclonal antibody. c Immunoblotting with the polyclonal antibody revealed that patients 1–5 with congenital TTP all lacked the ADAMTS13 band, whereas patients 6 and 7 with acquired TTP presented a normal expression pattern. Normal plasma (NP) was run on the same gel for comparison. d These results were confirmed by the monoclonal anti-ADAMTS13 antibody. e Immunoblot using the polyclonal antibody. Immunoadsorption of immunoglobulins from the plasma samples of patients 6 and 7 with acquired TTP lead to the simultaneous removal of the ADAMTS13 band, indicating that ADAMTS13 is complexed with the anti-ADAMTS13 auto-antibodies. In contrast, the ADAMTS13 band remained visible in normal plasma (NP) treated similarly. f A schematic presentation of the mechanism by which the removal of immunoglobulins leads to the removal of the ADAMTS13 antigen from the plasma of patients with acquired TTP. The plasma sample contains ADAMTS13 (filled circles), auto-antibodies to ADAMTS13 (⤙), and various other plasma proteins (open circles). Immunoblotting of the plasma sample prior to the removal of immunoglobulins detects the presence of ADAMTS13 antigen. Passage of the plasma sample through a protein G-sepharose column leads to the binding and removal of all immunoglobulins from the sample. Since ADAMTS13 is bound to the anti-ADAMTS13 auto-antibodies, it is removed along with them. Immunoblotting of the flow-through shows no ADAMTS13 band. g Immunoblot with the monoclonal antibody showing a normal ADAMTS13 band in the plasma of the parents, which are all heterozygous for one mutated allele and are clinically unaffected. Lane 1: the mother of patients 1 and 2, 4143insA; lane 2: the mother of patient 3, P353L; lane 3: the father of patient 3, P457L; lane 4: the mother of patient 4, P671L; lane 5: the father of patient 4, 4143insA. Normal plasma (NP) was run on the same gel for comparison
Detection of ADAMTS13 in plasma samples from TTP patients
The plasma samples from all patients with congenital TTP, regardless of the mutation, lacked the ADAMTS13 band (Fig. 1c). Patients 6 and 7 with acquired TTP revealed the same ADAMTS13 protein expression pattern as the controls (Fig. 1c). These results were obtained using both the polyclonal (Fig. 1c) and the monoclonal antibody (Fig. 1d). When immunoadsorption of plasma immunoglobulins was carried out prior to immunoblotting, the ADAMTS13 band remained visible in normal plasma, but was completely removed from the samples of the patients with acquired TTP (Fig. 1e), probably due to its association with the immunoglobulin inhibitor.
Detection of ADAMTS13 in the parents of the TTP patients
The parents of the TTP patients are all carriers of one ADAMTS13 mutation and one normal allele. All parents presented a normal ADAMTS13 phenotype using both the polyclonal (data not shown) and the monoclonal antibody (Fig. 1g).
Discussion
In the present study, we detected ADAMTS13 in plasma using a polyclonal and a monoclonal antibody. This assay was capable of distinguishing TTP patients from normal individuals, as well as differentiating between congenital and acquired TTP. Plasma from the patients with congenital TTP lacked the ADAMTS13 antigen. In contrast, the plasma of patients with acquired TTP expressed a normal ADAMTS13 phenotype.
Previous studies describing the ADAMTS13 phenotype in normal plasma by immunoblotting with other specific anti-ADAMTS13 antibodies have shown immunoreactive bands of the same molecular weight using similar conditions [33, 46]. The ADAMTS13 antigen in patients with congenital and acquired TTP has recently been shown by ELISA, demonstrating low to undetectable ADAMTS13 levels in patients with congenital TTP [11, 38] and decreased, but mostly detectable, levels in patients with acquired TTP [11, 38, 45]. In the present study, the ADAMTS13 phenotype in TTP patients is described by immunoblotting, confirming the lack of ADAMTS13 antigen in the plasma of patients with congenital TTP and the presence of circulating complexes in acquired TTP. Furthermore, we showed that heterozygous carriers of the ADAMTS13-related mutations who, thus, have reduced ADAMTS13 bioactivity have a normal phenotype.
The plasma of the patients with congenital TTP did not present the ADAMTS13 band. This may be due to altered synthesis, secretion or antigenicity, or due to increased breakdown of the protease in plasma. The fact that two antibodies directed to two different domains in ADAMTS13 were unable to detect the protease band makes altered antigenicity less likely to be the cause for the lack of the ADAMTS13 band in these patients. Previous studies have shown impaired secretion of the 4143insA (patients 1, 2, 4, and 5) [35, 42, 44] and P353L (patient 3) [42] mutants from cells, thus, indicating that the protease may accumulate intracellularly, at least in some patients with congenital TTP. This may be due to a missing cell sorting signal, as in the case of 4143insA [44] or due to conformational changes in the protein, impairing its secretion. Similar findings regarding two other ADAMTS13 mutations, V88M and G239V, have been recently reported [34]. A total lack of ADAMTS13 activity in the plasma is thought to be incompatible with life [27], thus, the patients may have very low amounts of ADAMTS13 activity in their plasma, which we were unable to detect with this method.
The patients with acquired TTP presented with a normal ADAMTS13 band, which is consistent with the fact that ADAMTS13 protease genotype and expression are normal, but their activity is lower, due auto-antibodies [11, 14, 41, 45, 52]. The finding that the immunoadsorption of immunoglobulins from the plasma of the patients with acquired TTP also led to the removal of the ADAMTS13 antigen from their samples indicates that the protease is complexed with the auto-antibodies in the circulation of these patients, and that this occurs even during clinical remission.
The majority of the ADAMTS13 assays available today detect ADAMTS13 activity levels in plasma (Table 2). This is performed either by detecting the VWF products resulting from ADAMTS13 cleavage (assays 1–5) or by measuring the residual VWF activity (assays 6–7). The VWF substrate utilized in these assays may be high molecular weight VWF (plasma-derived or recombinant; assays 1–4) or VWF domains or short synthetic peptides (assay 5). Two multicenter studies evaluating methods 1, 3–5, and 6–7 showed that all assays were able to detect severely ADAMTS13-deficient plasma samples and indicated that methods 1, 6, and 7 were the most consistent and reliable methods [48, 49]. A recent smaller study evaluating the FRETS-VWF73 method (assay 5) showed that this is a reliable assay which provides results in good accordance with other methods [26].
Table 2ADAMTS13 assaysASSAYPRINCIPLEREFERENCEDetection of ADAMTS13 activityDetection of VWF cleavage products 1. VWF multimer structure analysisDetection of the breakdown of high-molecular-weight VWF[15, 23] 2. Immunoblotting of VWF Detection of cleavage products of native VWF or recombinant VWF domains[37, 50] 3. IRMADetection of VWF cleavage products[32] 4. Flow assayDetection of the breakdown of ULVWF-platelet strings attached to endothelial cells[1] 5. Various methods using VWF domains or short synthetic VWF  peptides as the substrate, such as the FRETS-VWF73 assayDetection of cleavage products of the VWF domains or VWF peptides[9, 18, 22, 25, 59–61]Detection of VWF residual activity 6. Collagen bindingDetection of VWF binding to collagen; binding correlates to VWF multimer size[17] 7. Ristocetin cofactor activityDetection of platelet aggregates; VWF ability to induce platelet aggregates in the presence of ristocetin correlates to multimer size[7]Detection of ADAMTS13 antigen and auto-antibodies 8. ELISADetection of ADAMTS13 antigen or anti-ADAMTS13 auto-antibodies[11, 38, 39, 41, 54] 9. ImmunoblottingDetection of anti-ADAMTS13 auto-antibodies[57] 10. Present assay (immunoblotting)Detection of ADAMTS13 antigen and size and (indirectly) of auto-antibodies–Mutation analysis 11. PCRDetection of mutations in the ADAMTS13 gene[27]IRMA: immunoradiometric assay; FRET: fluorescence resonance energy transfer; FRETS-VWF73: a 73-amino-acid-long synthetic peptide which provides a minimal substrate for ADAMTS13 [24] that has been made fluorogenic; PCR: polymerase chain reaction
Few recent studies have shown that ADAMTS13 antigen was detectable in plasma by ELISA (assay 8 in Table 2). ELISA assays are a valuable complement to the ADAMTS13 activity assays, and offer a fast analysis of the ADAMTS13 antigen levels in plasma samples using antibodies directed to ADAMTS13. These assays are able to distinguish ADAMTS13-deficient TTP plasma from normal plasma samples. Other methods are capable of detecting anti-ADAMTS13 auto-antibodies (Table 2, assays 8–9). Mutational analysis can be carried out by polymerase chain reaction (PCR) in order to detect mutations in the ADAMTS13 gene (assay 11 in Table 2).
In the present study, we developed a qualitative, semi-quantitative assay capable of detecting ADAMTS13 antigen and anti-ADAMTS13 auto-antibodies in plasma and, thus, distinguishing between TTP and normal plasma, as well as distinguishing between congenital and acquired TTP. All patients with congenital TTP, regardless of their ADAMTS13 mutations, presented with undetectable levels of ADAMTS13 antigen, whereas patients with acquired TTP presented a normal phenotype. Heterozygous carriers of ADAMTS13 mutations also revealed a normal ADAMTS13 antigen band. Although not a quantitative method like the above-mentioned ELISA assays, the present assay offers the advantage of not only demonstrating the presence or absence of ADAMTS13 antigen, but also the molecular size of ADAMTS13 present in plasma samples. Furthermore, this assay was able to show the presence of anti-ADAMTS13 antibodies indirectly by performing immunoadsorption of plasma immunoglobulins prior to immunoblotting. In conclusion, this assay is able to distinguish between normal and TTP plasma, and also between congenital and acquired TTP. It is easy to perform and can be used at any hospital laboratory routinely using SDS-PAGE electrophoresis and immunoblotting, and offers a complement to existing ADAMTS13 methods in the diagnosis of TTP.

adamts13
plasma
thrombotic thrombocytopenic purpura
von willebrand factor
von willebrand factor cleaving protease
immunoblotting


Iron therapy for renal anemia: how much needed, how much harmful?

Iron deficiency is the most common cause of hyporesponsiveness to erythropoiesis-stimulating agents (ESAs) in end-stage renal disease (ESRD) patients. Iron deficiency can easily be corrected by intravenous iron administration, which is more effective than oral iron supplementation, at least in adult patients with chronic kidney disease (CKD). Iron status can be monitored by different parameters such as ferritin, transferrin saturation, percentage of hypochromic red blood cells, and/or the reticulocyte hemoglobin content, but an increased erythropoietic response to iron supplementation is the most widely accepted reference standard of iron-deficient erythropoiesis. Parenteral iron therapy is not without acute and chronic adverse events. While provocative animal and in vitro studies suggest induction of inflammation, oxidative stress, and kidney damage by available parenteral iron preparations, several recent clinical studies showed the opposite effects as long as intravenous iron was adequately dosed. Thus, within the recommended international guidelines, parenteral iron administration is safe. Intravenous iron therapy should be withheld during acute infection but not during inflammation. The integration of ESA and intravenous iron therapy into anemia management allowed attainment of target hemoglobin values in the majority of pediatric and adult CKD and ESRD patients.
Introduction
Iron-restricted erythropoiesis is a common clinical condition in patients with chronic kidney disease (CKD). The causes underlying this pathology and the subsequent contribution of absolute or functional iron deficiency to renal anemia include:
Inadequate intake of dietary ironBlood loss during the extracorporeal procedure in hemodialysis patientsBlood loss from the gastrointestinal tract (bleeding)(Too) frequent diagnostic blood testsInadequate intestinal iron absorption and inhibition of iron release from macrophages (anemia of chronic disease)Increased iron requirements during therapy with erythropoiesis-stimulating agents (ESAs).
In iron deficiency without anemia, reduction in iron storage is not sufficiently large enough to decrease the hemoglobin level. In CKD patients with absolute iron-deficient anemia, however, iron deficit is so severe that it aggravates renal anemia. Iron supplementation is mandatory in the majority of patients with end-stage renal disease (ESRD), particularly in those receiving ESA therapy.
Evaluation of iron status
In patients with normal kidney function, absolute iron deficiency is characterized by low serum ferritin concentration (<30 μg/l). The ferritin cut-off level for absolute iron deficiency in CKD patients is 100 μg/l [1] by the experience that chronic inflammation increases serum ferritin levels approximately three-fold. The Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines recommend serum ferritin levels >200 μg/l for the adult hemodialysis patient population [2]. The European Best Practice Guidelines define the optimal range for serum ferritin as 200–500 μg/l in adult patients with ESRD [1]. A normal ferritin level (≥100 μg/l) cannot exclude iron deficiency in uremic children [3, 4], but a serum ferritin <60 μg/l is a specific predictor of its presence [5]. An upper ferritin level of 500 μg/l is recommended for adults and children with CKD [2]. Serum ferritin is an indicator of storage iron. Iron deficiency is accompanied by reductions in serum iron concentration and transferrin saturation (TSAT) and by elevations in red cell distribution width, free erythrocyte protoporphyrin concentration, total serum iron binding capacity (TIBC), and circulating transferrin receptor [6]. Serum soluble transferrin receptor, however, reflects ongoing erythropoiesis but not iron availability in ESA-treated chronic dialysis patients [7]. Typically, TSAT (the ratio of serum iron to TIBC) is 15% or less (normal 16–40%) with iron deficiency, but TSAT also decreases in the presence of acute and chronic inflammation (functional iron deficiency). TSAT is raised with bone marrow dysfunction due to alcohol, cancer chemotherapy, or a megaloblastic process. TSAT is also affected by diurnal variations, being higher in the morning and lower in the evening [8]. Even a TSAT > 20% or a serum ferritin level > 200 μg/l does not exclude iron deficiency in ESRD patients. In a study by Chuang et al. [9], 17% of iron-deficient hemodialysis patients had serum ferritin levels greater 300 μg/l. Clinically, functional iron deficiency is confirmed by the erythropoietic response to a course of parenteral iron and is excluded by the failure of erythroid response to intravenous iron administration [10].
Erythrocyte and reticulocyte indices, such as the percentage of hypochromic red blood cells and the reticulocyte hemoglobin content (CHr) provide direct insight into bone marrow iron supply and utilization. Determination of the percentage of hypochromic red blood cells, i.e., those with a cellular hemoglobin concentration <28 g/dl, provides important information on functional iron deficiency in ESA-treated dialysis patients [11]. Tessitore et al. [12] found that hypochromic red blood cells >6% are the best marker to identify adult ESRD patients who will have the best response to intravenous iron. CHr has been proposed as a surrogate marker of iron status and as an early predictor of response to iron therapy in adult dialysis patients [13, 14]. Combined use of CHr and high-fluorescence reticulocyte count predicts with a very high sensitivity and specificity the response to intravenous iron in adult dialysis patients [8]. There are, however, only few studies in the pediatric renal literature on the use of CHr [15, 16]. In children with ESRD, an increase from baseline CHr levels was observed in response to oral and intravenous iron, but cut-off values for the use of CHr in the pediatric CKD population are not clear. This measure has proven to be of value with adult ESRD patients.
Detection of both absolute and functional iron deficiency is important because iron deficiency is the most common cause of hyporesponsiveness to ESAs. In clinical practice, an increased erythropoietic response to iron supplementation is the most widely accepted reference standard of iron-deficient erythropoiesis. For pharmacological therapy of iron deficiency, both oral and parenteral iron preparations are available. Intravenous iron is more effective than oral iron supplementation, at least in CKD patients. Iron is not only a prerequisite for effective erythropoiesis but also an essential element in all living cells. Elemental iron serves as a component of oxygen-carrying molecules and as a cofactor for enzymatic processes. Its redox potential, however, limits the quantity of iron that can be safely harbored within an individual.
Oral iron therapy
Oral iron is best absorbed if given without food. Side-effects of oral iron therapy include constipation, diarrhea, nausea, and abdominal pain. In the treatment of iron deficiency with ferrous sulphate, the usual adult dose is one 300 mg tablet (containing 60 mg elemental iron) three to four times daily. The pediatric dose is 2–6 mg/kg per day of elemental iron in 2–3 divided doses [17, 18]. Intestinal iron absorption is enhanced in patients with iron deficiency and declines with the correction of iron deficiency and reaccumulation of iron stores. If side-effects limit compliance, the medication can be administered with food, or the dose can be reduced. One 500-mg ferrous sulphate dose nightly at bedtime may be an effective therapy in adults [19].
Uremia is a chronic inflammatory state [20, 21]. Even in the absence of overt infection or inflammation, many ESRD patients show increased levels of acute-phase proteins, such as C-reactive protein (CRP), ferritin, fibrinogen, and/or interleukin-6 (IL-6), associated with low serum albumin levels [22]. The interaction of proinflammatory cytokines with hepcidin in mediating functional iron deficiency may explain why CKD patients have high ferritin levels, poor intestinal iron absorption, and disturbed iron release from the reticuloendothelial system [23].
In the duodenum and proximal jejunum, the nonheme dietary Fe3+ is reduced to Fe2+ by the cytochrome b-like ferrireductase Dcytb. Fe2+ is gathered from the lumen of the intestine and crosses the apical enterocyte brush border membrane through the divalent metal transporter-1 (DMT1). The expression of both Dcytb and DMT1 is strongly affected by the iron concentration within the enterocyte. Circulating levels of hepcidin negatively regulate intestinal iron absorption by the enterocyte DMT1. Hypoxia, anemia, iron deficiency, and/or stimulated erythropoiesis strongly down-regulate hepatic hepcidin release, allowing intestinal iron absorption, while iron overload or inflammation/infection stimulates hepcidin production, resulting in inhibition of intestinal iron absorption. Hepcidin controls the whole-body iron content. It also inhibits the release of iron by the iron exporter ferroportin (iron-regulated transporter-1) located along the entire basolateral membrane of enterocytes and also in the intracellular vesicular compartment of tissue macrophages. Hepcidin is primarily produced in the liver in response to acute-phase reactions. Any further expression depends on the degree of hepatic iron storage (for review, see [24]). Thus, the inflammatory state associated with uremia and less uremia per se is predominantly responsible for poor intestinal iron absorption in ESRD patients. In iron-depleted peritoneal dialysis patients with normal CRP values, high-dose oral iron is well absorbed [25].
The European Pediatric Peritoneal Dialysis Working Group recommended that anemia treatment should aim for a target hemoglobin concentration of at least 11 g/dl accomplished by administration of ESA and iron. Oral iron should be preferred in pediatric peritoneal dialysis patients [18]. The majority of pediatric hemodialysis patients are also supplemented with oral iron. The 2001 North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) annual report showed that 84% of pediatric peritoneal dialysis and 72% of pediatric hemodialysis patients at 12 months of dialysis were receiving oral iron therapy [26].
Intravenous iron therapy
Since oral iron therapy is often not sufficient in ESRD patients, parenteral administration of iron is necessary to optimally care for these patients. Intravenous iron can be given safely to CKD patients [27–34] as long as the therapy is performed according to international recommendations and guidelines [1, 2]. This therapy is unequivocally superior to oral iron supplementation [35]. All forms of intravenous iron may be associated with acute adverse events [1, 2]. Potential risk factors associated with intravenous iron therapy include acute allergic reactions such as rash, dyspnoea, wheezing, or even anaphylaxis, as well as long-term complications caused by the generation of powerful oxidant species, initiation and propagation of lipid peroxidation, endothelial dysfunction, propagation of vascular smooth muscle cell proliferation, and/or inhibition of cellular host defense. Allergy is believed to relate to dextran moiety. Iron dextran therapy is associated with a higher risk for serious type I reactions compared with newer intravenous iron products. Iron sucrose carries the lowest risk for hypersensitivity reactions [36]. In our clinical experience with more than 100,000 intravenous injections of iron sucrose and ferric gluconate within the last 15 years, we detected no significant differences in efficacy or adverse events between both intravenous iron preparations. Serious reactions to iron dextran are unpredictable and possibly life threatening. Labile- or free-iron reactions are more frequent with nondextran forms [37]. Recommended doses of iron sucrose or ferric gluconate appear safe, at least in adult CKD patients [32, 34, 38]. Parenteral therapy with iron sucrose or ferric gluconate is also safe and effective in the management of anemia in adult hemodialysis patients sensitive to iron dextran [29, 39]. Iron sucrose safety data are sparse in the pediatric CKD literature [2]. It should be considered that the iron load administered intravenously to CKD patients based on international recommendations is more than ten times less than the iron load by repeated blood transfusions at times when no ESA therapy was available for ESRD patients.
Iron deficiency in CKD patients develops primarily during the correction of renal anemia by ESA treatment. Approximately 150 mg of iron is necessary for an increase of 1 g/dl in hemoglobin level. In adult hemodialysis patients, annual blood losses up to 4 l of blood, equivalent to 2 g iron, should be considered [40]. Thus, intravenous iron prevents iron-restricted erythropoiesis during ESA therapy. Parenteral treatment strategies depend on the availability of iron products in respective countries. Hemodialysis patients should receive at least one dose of intravenous iron every 2 weeks [1]. Careful monitoring of iron status is mandatory in order to avoid iron overload. In patients with anemia of chronic disease (and inflammation), a major part of iron administered intravenously is transported into the reticuloendothelial system, where it is not readily available for erythropoiesis [41].
Intravenous iron therapy is underused in pediatric ESRD patients. Chavers et al. [42] compared anemia prevalence in US Medicare pediatric and adult dialysis patients treated with ESAs from 1996 to 2000. Prevalence of anemia (defined as hemoglobin values less than 11 g/dl) was found in pediatric and adult hemodialysis patients during 54.1% versus 39.8% patient years as well as in pediatric and adult peritoneal dialysis patients during 69.5% versus 55.1% patient years, respectively. The percentage of patient years with intravenous iron was low, especially for pediatric peritoneal dialysis patients: 33.9% (age group 0–4 years) and 71% (age group 5–19 years) versus 0.3% and 19.4% among pediatric hemodialysis patients in these age categories, respectively. Among pediatric hemodialysis and peritoneal dialysis patients, intravenous iron was not administered among 34% and 85% of patient years [42]. Data obtained from the US Centers for Medicare and Medicare Services on hemodialysis patients in an age range between 12 and <18 years indicate that 37% of these patients are anemic, defined as hemoglobin <11 g/dl. Dialyzing <6 months, a low serum albumin, and a mean TSAT <20% were identified as predictors of anemia in these children. Despite the prescription of iron supplements in almost all pediatric patients, there was evidence for low TSAT and/or low ferritin in many children. In this study, approximately 60% of all children received intravenous iron therapy [43].
An international multicenter study investigated the safety and efficacy of two dosing regimens (1.5 mg/kg or 3 mg/kg) of ferric gluconate during eight consecutive hemodialysis sessions in iron-deficient pediatric hemodialysis patients receiving concomitant ESA therapy. Efficacy and safety profiles were comparable, with no unexpected adverse events with either dose [16]. Initial recommended ferric gluconate therapy is 1.5 mg/kg for eight doses for iron-deficient pediatric hemodialysis patients and 1 mg/kg per week for iron-replete pediatric hemodialysis patients, with subsequent dose adjustments made according to TSAT and/or ferritin levels [16, 44]. In children, iron sucrose in a high dose (5 mg/kg) should be given over 90 min and in a low dose (up to 2 mg/kg) over 3 min [45]. An other recommendation for pediatric patients is to inject 6 mg iron/kg per month during iron deficiency, with subsequent dose adjustments according to serum ferritin [46]. Nonrandomized intravenous iron (1–4 mg/kg per week) trials in children on hemodialysis and nondialyzed or transplanted children showed an increase in hemoglobin or hematocrit and a decrease in ESA requirements between 5% and 62% per week or per dose of ESA [3, 4, 47, 48]. De Palo et al. [49] reported an excessive increase in hemoglobin with severe hypertension in children on maintenance hemodialysis after the first month of darbepoetin alpha therapy combined with intravenous ferric gluconate in a very high dose of 10–20 mg/kg per week. The patients had already been on erythropoietin therapy for at least 6 months with adequate iron status (serum ferritin 220 ± 105 μg/l; TSAT 24.2 ± 11.5%). The complications observed in this study are not surprising, as such a high-dosed intravenous iron therapy is not justified, either in adult or in pediatric dialysis patients with “adequate iron status”. The current practice of intravenous iron therapy in pediatric hemodialysis patients is often performed on extrapolation from adult data and not based on data obtained from prospective multicenter trials performed in children [50].
Even if exposure to intravenous iron may lead to oxidative stress, renal injury, infection, and/or cardiovascular disease, the magnitude of these complications is not really clear. The overall risk–benefit ratio favors the use of intravenous iron in CKD patients in order to optimize erythropoiesis and prevent iron deficiency [51]. Intravenous iron therapy is still underutilized in the adult hemodialysis population [52], but its use increased from 1997 to 2002. Ferric gluconate and iron sucrose have become the predominant form of intravenous iron therapy [53].
Iron and inflammation/infection
Intravenous iron therapy may adversely impact CKD patients via a potentiation of systemic inflammation. In animals, even a single ultra-high-dosed intravenous injection of available iron preparations (2 mg iron for mice with a body weight of 25–35 g, corresponding to 5,000 mg iron for a 75-kg adult patient) does not independently raise plasma levels of tumour necrosis factor-α (TNF-α). Systemic inflammation experimentally induced by intraperitoneal endotoxin injection (2 or 10 mg/kg in mice) resulted in a dramatic increase in plasma TNF-α levels. Interestingly, 2 h following concomitant injection of endotoxin and ferric gluconate (2 mg) or iron dextran (2 mg), a decrease of plasma TNF-α levels was observed. In contrast, combined endotoxin and iron sucrose injection resulted in a further increase in plasma TNF-α compared with endotoxin alone [54]. However, a 75-kg CKD patient will neither receive 5,000–25,000 mg endotoxin intraperitoneally nor concomitant 5,000 mg iron sucrose injection intravenously in order to demonstrate that under these artificial conditions, TNF-α mRNA and TNF-α release are stimulated. It is therefore of particular importance that relevant clinical studies demonstrated that intravenous iron sucrose therapy within recommended doses may even display anti-inflammatory effects [55, 56].
Intravenous iron sucrose therapy affects positively circulating cytokine levels in hemodialysis patients: IL-4 levels increase, while TNF-α levels decrease [55]. There is a direct correlation between IL-4 and TSAT but an inverse correlation between TNF-α and TSAT. Hemoglobin levels increase with an increase of IL-4 and a decrease of TNF-α, while ESA dose decreases with an increase of IL-4 and a decrease of TNF-α [55]. In other words, adequately dosed intravenous iron therapy in hemodialysis patients results in down-regulation of proinflammatory immune effector pathways and stimulation of the expression of the anti-inflammatory cytokine IL-4. By these mechanisms, in addition to its well-known stimulatory effects on erythropoiesis, iron therapy contributes to an increase in hemoglobin levels and to a decrease in the need of ESAs. The anti-inflammatory properties of intravenous iron therapy have also been demonstrated in patients with rheumatoid arthritis [56].
In contrast, iron-mediated weakening of the Th-1 immune effector function (estimated by lowered TNF-α production) with a subsequent strengthening of Th-2-mediated immune effector function (estimated by increased IL-4 production) is an unfavorable condition for ESRD patients in the case of an acute infection or malignant disease [55]. Moreover, intravenous administration of iron increases the availability of this essential nutrient for microorganisms [57] associated with an increased incidence of infectious complications in ESRD patients. Teehan et al. [58] followed 132 hemodialysis patients for up to 1 year after the initiation of intravenous iron therapy for the outcome of bacteremia. Iron-replete patients (those with a TSAT value ≥ 20% and a ferritin level ≥ 100 ng/ml) had a significantly higher risk of bacteremia (hazard ratio 2.3 in the univariate analysis and 2.5 in the multivariate analysis) compared with adult hemodialysis patients who were not iron replete [58]. Inhibition of intracellular killing of bacteria by polymorphonuclear leukocytes (PMNL) due to iron sucrose therapy in high-ferritin hemodialysis patients has been reported [59]. Peritoneal dialysis patients receiving high-dose intravenous iron sucrose also displayed short-term inhibition of bacterial killing by PMNL [60]. Finally, iron sucrose as well as ferric gluconate inhibit in vitro migration of PMNL through endothelial cells [61]. All these data suggest a risk for infectious complications, at least in patients overtreated with iron.
However, clinical studies on intravenous iron therapy in ESRD patients reported controversial results [62–64]. According to the published cohort study, among 32,566 hemodialysis patients, there was no association between iron administration and mortality. This study by Feldman and coworkers [64] supports intravenous administration of iron ≥1,000 mg over 6 months if needed to maintain target hemoglobin levels. This is, however, an adult and not a pediatric recommendation. Intravenous iron therapy should be withheld in the presence of acute infection until the infection has successfully been treated and resolved [65]. Intravenous iron is ineffective and may increase the virulence of bacterial and viral pathogens. On the other hand, ESRD patients with chronically infectious complications may develop absolute iron deficiency if iron supplementation is withheld over months. In such a situation, iron should be administered intravenously as soon as ferritin levels drop below 100 μg/l (personal opinion). ESA-stimulated erythropoiesis of chronically infected adult ESRD patients may benefit from low-dose intravenous iron supplementation (10–20 mg iron sucrose or ferric gluconate per hemodialysis session), even if serum ferritin is normal or slightly elevated. The level of serum ferritin at which ESRD patients are considered to be in an iron overload state is still not defined. Inflammatory states should not be considered indiciations to withhold the benefits of intravenous iron therapy in general [65]. However, a clinical problem is the diagnosis of chronic anemia associated with inflammation and true iron deficiency, as serum ferritin concentration increases rather than decreases.
Iron and kidney function
Intravenous administration of 100 mg iron sucrose in CKD patients caused transient proteinuria and tubular damage [37], but ferric gluconate did not (125 mg infused over 1 h or 250 mg infused over 2 h) [66]. Induction of passive Heymann nephritis in rats resulted in a marked increase in nonheme iron content of kidney cortex and tubules, while a iron-deficient diet caused a significant reduction of nonheme iron level in glomeruli and also a significant reduction of proteinuria in these animals [67]. Pediatric thalassemia patients have a high prevalence of renal tubular abnormalities, probably caused by the anemia and increased oxidative stress induced by excess iron deposits. Significantly higher levels of urinary N-acetyl-beta-d-glucosaminidase, malondialdehyde, and beta-2-microglobulin were found in these children compared with normal children [68]. Under artificial experimental conditions (intravenous injection of 2 mg iron sucrose or ferric gluconate into mice with a body weight of 25–35 g), induction of monocyte chemoattractant protein-1 (MCP-1) in renal and extrarenal tissues has been observed. Since MCP-1 has profibrotic properties, implications for CKD progression in case of intravenous iron therapy has been suggested [69]. However, in a recent article, Mircescu et al. [70] reported that intravenous iron sucrose therapy (200 mg elemental iron per month for 12 months) resulted in an increase in hemoglobin from 9.7 ± 1.1 to 11.3 ± 2.5 g/dl in nondiabetic patients with CKD and a mean glomerular filtration rate (GFR) of 36.2 ± 5.2 ml/min per 1.73 m2, estimated by the formula of Cockcroft and Gault. The majority of these CKD patients had preexisting iron deficiency (mean ferritin 98.0 μg/l, range 24.8–139.0 μg/l). An important finding of this study was that GFR (final values at the end of the study 37.2 ± 0.9 ml/min per 1.73 m2) remained completely stable over a period of 12 months despite 2,400 mg of intravenous iron sucrose administration. The CKD patients had relatively high blood pressure (140 ± 32/82 ± 20 mmHg at baseline), which did not change throughout the investigation [70]. Agarwal [71] found that a single dose of 100 mg iron sucrose results in a transient increase of MCP-1 in plasma and urine of CKD patients. Those who believe that 100 mg iron sucrose administered to CKD patients may negatively affect kidney function should simply administer a lower intravenous iron dose, e.g., 50 mg iron sucrose intravenously.
Iron and oxidative stress
Zager et al. [72] compared in vitro parenteral iron toxicity induced by three commercially available iron preparations (iron dextran, ferric gluconate, iron sucrose) using renal tubular cells and renal cortical homogenates. Each test agent induced massive and similar degrees of lipid peroxidation. Under the in vitro conditions used, iron sucrose caused markedly higher cell death than ferric gluconate, and ferric gluconate caused higher cell death than iron dextran. This relative toxicity profile was also observed in cultured aortic endothelial cells. Again, it should be stressed that the study of Mircescu et al. [70] demonstrated that intravenous iron sucrose therapy administered within international recommendations (none of the 58 CKD patients exceeded serum ferritin of 500 μg/l) does not cause a decline in kidney function in CKD patients over a period of 1 year.
Intravenous iron therapy may enhance symptoms of oxidative stress [73–75]. Drüeke et al. [75] demonstrated that advanced oxidation protein products (AOPPs) correlated with iron exposure and carotis artery intima thickness in dialysis patients. In hemodialysis patients, oxidative stress as a result of intravenous iron therapy caused serum albumin oxidation [76]. Ferric gluconate modifies fibrinogen and β2-microglobulin as a marker of oxidative stress in adult hemodialysis patients [77, 78]. Intravenous administration of 100 mg iron sucrose in CKD patients increased malondialdehyde as a marker of lipid peroxidation [37]. Hemodialysis patients with ferritin levels above 650 μg/l showed an enhanced oxidative burst in PMNL [59]. However, not all studies found evidence for enhanced oxidative stress caused by parenteral iron therapy in ESRD patients. Hemodialysis therapy per se was found to cause a significant increase in peroxide concentration. Interestingly, this rise in plasma total peroxides was not additionally influenced by concomitant intravenous injection of 100 mg iron sucrose [79]. These data confirm increased oxidative stress associated with hemodialysis [80]. Whether intravenous iron therapy results in an additional oxidative stress reaction needs to be further evaluated.
Increased blood levels of non-transferrin-bound iron (NTBI) and/or its redox-active part have been reported in adult ESRD patients receiving intravenous iron therapy [79, 81, 82]. Intravenous infusion of 300 mg iron sucrose in ESRD patients also caused peripheral vasodilation, which was confirmed by increased forearm blood flow. NTBI and redox-active iron were considered to be, at least in part, responsible for endothelial dysfunction observed in ESRD patients. However, an increase in NTBI and redox-active iron caused by intravenous iron sucrose infusion did not influence vascular reactivity to intra-arterial acetylcholine, glycerol-trinitrate, or L-N-mono-methyl-arginine (L-NMMA) [82].
Vitamin C and iron
ESRD patients undergoing regular hemodialysis or hemodiafiltration may develop vitamin C deficiency [83]. Vitamin C deficiency may cause oxidative stress and vascular complications as well as impairment of intestinal iron absorption and iron mobilization from iron stress. Moretti et al. [84] measured iron absorption in young women from test meals fortified with isotopically labeled ferric pyrophosphate and ferrous sulfate. The addition of ascorbic acid at a molar ratio of 4:1 to iron increased iron absorption from ferric pyrophosphate to 5.8% and that from ferrous sulfate to 14.8%. In the fasting state, ferrous ascorbate is better absorbed than ferric hydroxide-polymaltose complex [85]. High-dose oral vitamin C may increase intestinal aluminium absorption [86]. Oxidative stress can cause hyporesponsiveness to ESA therapy in ESRD patients. Vitamin C may improve erythropoiesis through its antioxidative properties [87]. Intravenous ascorbic acid therapy facilitates iron release from inert deposits, resulting in a decrease of soluble transferrin receptor and an increase of TSAT [88]. In contrast, oral vitamin C supplementation (250 mg three times per week for 2 months) did not influence oxidative/antioxidative stress and inflammation markers in adult hemodialysis patients [89].
In adult hemodialysis patients on maintenance intravenous iron sucrose therapy, intravenous administration of 500 mg ascorbic acid three times a week for 6 months resulted in an increase of TSAT and hemoglobin in approximately 65% of the patients [90]. In contrast, neither oral nor intravenous ascorbic acid changed TSAT or hemoglobin levels in a study performed by Chan et al. [91]. Ascorbic acid increases the intracellular labile iron pool and iron mobilization to transferrin in human hepatoma HepG2 cells only in the presence of iron sucrose but not in the presence of iron dextran or ferric gluconate [92]. Several studies reported an increase in hemoglobin and/or a decrease in adult ESA dose during adjuvant ascorbic acid therapy three times per week in ESRD patients [93–99]. Measurements of plasma oxalate concentration are needed in ESRD patients supplemented with ascorbic acid [100]. Studies on vitamin C and iron in children with ESRD are not available so far.
Iron therapy after kidney transplantation
Anemia is observed in 21–39.7% of adult renal transplant patients [101–104]. The prevalence may be even higher in pediatric transplant recipients: 84.3% of children were anemic in the first month after kidney transplantation, and prevalence of anemia was not below 64.2% between 6 months and 6 years after transplantation. Iron deficiency was identified in 27–56% of children between 1 and 60 months posttransplantation [105]. Fourteen pediatric and young adult renal transplant recipients received single iron gluconate infusions ranging from 1.9 to 6.4 mg/kg. The mean hemoglobin level increased significantly from 10.1 ± 1.6 to 11.4 ± 2.1 g/dl following ferric gluconate therapy. Adverse events were observed in three children [106]. A recent study by Morii and coworkers showed that oral coadministration of ferrous sulphate markedly decreased the absorption of mycophenolate mofetil in healthy Japanese subjects [107]. However, a randomized crossover trial failed to confirm this observation in European transplant patients receiving long-term mycophenolate mofetil therapy [108]. In line with this observation, an in vitro study showed that iron ions did not interact with mycophenolate mofetil [109].
Conclusions
The integration of ESA and intravenous or oral iron therapy into standard anemia management resulted in target hemoglobin levels (as established by international guidelines) in the vast majority of ESRD patients [110]. Correction of renal anemia reduced morbidity and mortality as well as hospitalization in ESRD patients. It also improved quality of life, cognitive function, and physical activity. Using a balanced approach to iron supplementation within international recommendations allowed the attainment of benefits of intravenous iron therapy at storage iron levels far below those generally seen with transfusions in the pre-ESA era [110].

erythropoiesis-stimulating agents
iron supplementation
iron status
inflammation
infection