Journal of Alzheimer's Disease 5 (2003) 189–195

Apolipoprotein E genotyping as a potential biomarker for mercury neurotoxicity

Michael E. Godfrey, Damian P. Wojcik, and Cheryl A. Krone

Abstract.

Apolipoprotein-E (apo-E) genotyping has been investigated as an indicator of susceptibility to heavy metal (i.e., lead) neurotoxicity. Moreover, the apo-E epsilon (ε)4 allele is a major risk factor for neurodegenerative conditions, including Alzheimer's disease (AD).

A theoretical biochemical basis for this risk factor is discussed herein, supported by data from 400 patients with presumptive mercury-related neuro-psychiatric symptoms and in whom apo-E determinations were made. A statistically relevant shift toward the at-risk apo-E ε4 groups was found in the patients (p < 0.001).

The patients possessed a mean of 13.7 dental amalgam fillings and 31.5 amalgam surfaces. This far exceeds the number capable of producing the maximum identified tolerable daily intake of mercury from amalgam. 

The clinical diagnosis and proof of chronic low-level mercury toxicity has been difficult due to the non- specific nature of the symptoms and signs. Dental amalgam is the greatest source of mercury in the general population and brain, blood and urine mercury levels increase correspondingly with the number of amalgams and amalgam surfaces in the mouth.

Confirmation of an elevated body burden of mercury can be made by measuring urinary mercury, after provocation with 2,3,-dimercapto-propane sulfonate (DMPS) and this was measured in 150 patients.

Apo-E genotyping warrants investigation as a clinically useful biomarker for those at increased risk of neuropathology, including AD, when subjected to long-term mercury exposures. Additionally, when clinical findings suggest adverse effects of chronic mercury exposure, a DMPS urine mercury challenge appears to be a simple, inexpensive procedure that provides objective confirmatory evidence. An opportunity could now exist for primary health practitioners to help identify those at greater risk and possibly forestall subsequent neurological deterioration

Introduction

Chronic exposure to mercury results in an accumulation in the brain, heart and kidneys, the main target organs [11,30,35]. Dental amalgam has been identified as the largest source of mercury vapor in the nonindustrially exposed population [49] and this vapor easily penetrates the central nervous system [34].

Amalgam is uniquely situated in the mouth with mercury having direct access to the olfactory lobes and the limbic brain, via the oro-nasal mucosa and via retrograde axonal transport of mercury, with subsequent preferential accumulation of mercury in those areas [4,19, 31,45,48]. Furthermore, brain mercury levels increase with the number of amalgam fillings or the amalgam "score" that is based on the number of dental surfaces covered in amalgam [31]. A recent US. NIH-funded study of military personnel also confirmed that blood and urine mercury levels corresponded to the number of amalgam fillings, averaging 4.5 times that of controls without amalgam [20]. These authors concluded that amalgam was a major, if not the main, source of mercury found in the body.  

At present, there are no readily acceptable in vivo diagnostic criteria for chronic mercury toxicity; diagnosis is predominantly based on patient history and clinical findings [35].

Long-term exposure to mercury results in central nervous system and other effects. These include: chronic fatigue, irritability, mood swings, poor concentration, mental confusion, chronic headaches, insomnia, and tremors [13,26]. A diagnosis of chronic low-level mercury toxicity, therefore, has been controversial both due to the non-specific nature of the symptoms and signs, and the lack of a reliable in vivo biomarker.

However, Apolipoprotein-E (apo-E) genotyping generally utilized in characterization of lipid metabolism, has been studied as a bio-indicator of susceptibility for Alzheimer's Disease (AD) [21,38– 40] and a recent paper on apo-E, lead exposure and neurobehavioral function has added support to an association with heavy metal intoxication [44]. A significant relationship was found between the apo-E epsilon (ε)4 allele, impaired cognitive function and susceptibility to toxicity in those previously chronically exposed to lead [44].

Apo-E genotyping determines the inherited parental epsilon 2, 3 or 4 groups, with six homozygous and heterozygous combinations being found (i.e., ε2/2, 2/3, 2/4, 3/3, 3/4 or 4/4). Isomer ε2 has two cysteine amino-acids in its structure, ε3 has one cysteine and one arginine, and ε 4 has two arginine amino-acids and no cysteine [6]. Cysteine, with its sulphydryl (-SH) bonds, is potentially able to bind to, and remove metals (e.g., mercury and lead) from tissues, whereas arginine, lacking the -SH bonds, would be unable to do this. 

Apo-E genotyping therefore becomes relevant once it is acknowledged that prolonged exposure to mercury has been associated with neurotoxicity, including the pathological histology unique to Alzheimer's senile dementia, namely, fibrillary tangles, amyloid plaques and increased phosphorylation of tau protein [12,27,28,32].

A complementary means for detection (and treatment) of heavy metal accumulation is the use of 2,3- dimercaptopropane-sulfonate (DMPS). Although DMPS had previously been extensively reviewed in Soviet literature, it was not until 1983 that a paper by Aposhian [1], detailing the history, pharmacology and uses of DMPS appeared in English.

Lead intoxication has been routinely treated for 20 years with the use of DMPS chelation. In 1989, a German toxicologist proposed using DMPS as a diagnostic test for chronic mercury retention and toxicity [9]. Either a 10-fold increase between the pre- and post-challenge urinary mercury levels or a post-challenge level greater than 50micrograms(μg), were suggested as indicators of an abnormally raised mercury body burden [9]. Both intravenous and orally administered DMPS are reported to be effective therapeutic agents [7], and useful in vivo biomarkers [3,9,17,29,42] for mercury accumulation.

DMPS is a better biomarker for low-level mercurialism than unchallenged urinary mercury excretion [2,3]. This paper discusses the use of apo-E genotyping and DMPS "challenge" testing as diagnostic adjuvants for identifying those at greater risk ofmercury intoxication. We also discuss a theoretical chemical basis for the observation [40] that the apo-E4 allele is a major risk factor for neurodegenerative conditions, including AD.

Discussion

There are three apo-E alleles (ε2, ε3, and ε4) that are universally distributed in the population with about 30% of the population carrying an ε4 allele [21,39]. Prior lipid research in New Zealand has indeed confirmed this distribution in New Zealanders with 27.2% of 426 blood donors having the ε4 isomer and 1% possessing ε4/4 [46]. Our cohort of patients with suspected mercury-associated symptomology exhibited a shift toward relatively higher proportions of ε4 isomers. 

For example, 34% of our subjects exhibited the combined 3/4 and 4/4 isomers compared to 26% found in (presumed healthy) blood donors [46], 23% in healthy Caucasian controls [39], 19% in previously exposed lead workers [44] and 14% in unselected populations [21]. The proportion of homozygous ε4/4 in our subjects was significantly greater (p < 0.001) than in the New Zealand blood donors (3.4% versus 1%). Furthermore, only 9.75% of our cohort had the reportedly AD-protective ε2/2, ε2/3 isomers compared to 21.4% in the latter and 17.7% in the general Australian population [25]. Our subjects resembled the relatively lower combined ε2/2 plus ε2/3 pattern in symptomatic leadexposed subjects (10.6% [44]).

These results suggest that apo-E genotyping could be used as a biomarker for identifying individuals more prone to experience mercury-related symptoms.

The susceptibility to the long-term effects of lead exposure on the central nervous system (CNS) has been associated with apo-E genotype. Stewart et al. [44] measured tibia bone lead levels and related these to neurobehavioral test scores and apo-E genotype. The authors concluded that the persistent CNS effect of lead may be more toxic in individuals who have at least one apo-E ε4 allele. To confirm the suspected elevated mercury levels in our cohort of symptomatic subjects, 150 patients consented to undertake DMPS urine mercury challenges.

The mean post-challenge mercury level in these patients of 347 μg/g creatinine was 9 times greater than the mean post-challenge mercury level of 39 μg reported in 10 asymptomatic controls (mean age 48 years) never exposed to amalgam [17].

The postchallenge mercury level in our subjects was circa 70 times greater than the pre-challenge mercury (∼5 μg/g creatinine). Aposhian et al. [2] reported a 25-fold increase in urinary mercury of amalgam bearers in the 9 hours after oral DMPS (17.2 μg total mercury excreted) compared to the 9 hours pre-DMPS (0.70 μg). Our higher percentage increase may be due to the rapid excretion following the IV 250 mg DMPS dose and the shorter collection time. If a longer collection is used (i.e., 6 hours), our urinary mercury concentration drops because most DMPS has been excreted within the first 90 minutes [17]. Dental personnel also exhibit raised urine mercury after oralDMPS (300mg p.o. after an 11hour fast) [18]. Six hour urine collections, before and after DMPS, revealed mercury levels and percentage increases generally similar to ours.

Our choice of the intravenous method of DMPS administration has an obvious time advantage for patients traveling from a distance. As with Aposhian et al. [2,3], the purpose of our test was intended to confirm a raised body burden and we were not primarily concerned with the absolute amount of mercury excreted. The test adds laboratory evidence to support the clinical differential diagnosis. Notwithstanding the small numbers so far involved, it was evident that irrespective of the apo-E genotype, those patients with larger amounts of amalgam or who had other metals in the mouth that potentially increased electrolysis, usually had considerably higher post-DMPS urine mercury levels. 

According to a 1968 New Zealand Health Department Survey of young people, those 15 years old averaged 13 amalgam fillings and those 21 years old averaged 16 fillings at that time [5]. Changes to dental practice were instigated in 1976, including a directive to cease prophylactic placement of amalgam into non-carious children's teeth. This resulted in a nationwide 64% reduction in amalgam fillings placed over the next 5 years [10] i.e. to < 8 fillings. However, there still remains the now middle-aged cohort of 1.4 million people currently between the ages of 35 and 65 years, out of a population of 3.8 million (NZ population statistics, June, 2000), who have many amalgam filled teeth. The 1996 Health Canada's Risk Assessment on  mercury vapor from amalgam, determined that a 70 kg adult would have reached a maximum "tolerable daily intake" (TDI) with 4 average-sized fillings or 8 dental surfaces of amalgam [37]. Based on this TDI, many of the currently middle-aged people with 30 surfaces of dental amalgam, could potentially be at an increased risk of toxicity, including AD. On average, our patients had greater than 3 times the number of fillings and amalgam surfaces that are associated with the maximum TDI. This mercury burden is consistent with the symptom prevalence in our patient cohort. 

Although apo-E genotyping has been predominantly directed towards lipid metabolism, it has been identified as a potential biomarker for AD senile dementia, with ε2 being protective and ε4 being predictive at a 70% level [38]. This association has been confirmed for both early and late onset AD in various ethnic populations [21]. However, ethnic variation in AD risk has been observed [21]. For example, in Japanese populations, the association of AD with the ε4 allele is much stronger in Caucasians. And, the AD risk in certain indigenous ethnic populations (e.g., some Africans) is much lower than might be expected from the relatively high frequency of ε4. It has been suggested that lifestyle factors (e.g., diet) may interact with apo-E genotype in raising the risk for ε4-associated disorders [8] such as coronary artery disease (CAD) and Alzheimer's Disease (AD).

The absence of the association of ε4 allele with CAD and AD in Sub-Saharan Africans, and its presence in African-Americans is consistent with this hypothesis. When ethnic populations with a high ε4 frequency shift from a traditional diet to the western dietary pattern, the frequency of these disorders increases [8]. We suggest that environmental toxicants, such as mercury from amalgams, could also be considered as a contributor to the changing risk when indigenous populations abandon their traditional, and adopt modern, lifestyles. 

According to Saunders, the underlying reason for the apo-E-associated differences in AD susceptibility remains a mystery [40]. However, a logical biochemical explanation has been proposed by Pendergrass and Haley, based on the different amino-acid configurations of the three apo-E isomers and their potential relevance to mercury elimination [33]. Only ε2 (with two cysteine -SH groups), and to a lesser extent ε3 (with one -SH group), are able to bind and remove mercury from the brain and cerebrospinal fluid. This would oppose accumulation of mercury which is reported to be causal for the unique brain lesions that typify the AD brain including neuro-fibrillary tangles [22,32,34].

We did not find any evidence of higher post-DMPS mercury excretion in those carrying the ε4 allele when compared to the other groups. However, this would not be unexpected if mercury from amalgam has direct access to the brain [4,19,31]. Mercury excretion after DMPS challenge reflects a general body burden and especially a renal loading [2,3]. Therefore, a DMPS challenge would not necessarily be a quantitative indicator of brain mercury levels.

Another aspect of AD pathology is the evidence that enhanced mitochondrial damage occurs in AD and ε4 genotype [16]. Mercury is very destructive at the mitochondrial level where catalase can demethylate organic mercury species into highly reactive inorganic mercury. Inorganic mercury is also an extremely potent enzyme inactivator [47]. Furthermore, chronic micro-mercurial toxicity specifically from dental amalgam has been documented  [14,23,31,36,45] and successfully treated by removal of amalgam and medical detoxification in 796 patients [23].

Still, not all research results agree with mercury's causal role in AD. Elevated mercury was not found in seven different regions of AD brains compared to controls [15]. However, the "controls" had possessed three amalgam surfaces whereas the AD subjects had six, likely obscuring any differences.

Therefore, based on the suggestion that mercury is causal for AD-like lesions due to its unique neurotoxicity, and that ε2 is potentially protective due to the cysteine amino-acids, it could be contended that: (i) those individuals who have inherited the ε4 allele and who are exposed to mercury, including from numerous dental amalgam fillings, would be at greater risk of developing AD at an earlier age than those with the ε2 configuration; and (ii) those with ε4/4 would potentially be at the greatest risk of accumulating mercury and developing symptoms.

Consistent with this, are our findings that short-term memory loss was common among our patients in the ε4 groups with only one of those possessing ε4/4 not listing this as a complaint. 

Two patients (both ε3/4) were already suffering from AD and there was a family history of some members having had senile dementia in 20% of the ε3/4, 4/4 cohorts.

Conclusion

The diagnosis of chronic mercury toxicity has traditionally been based on patient history and clinical acumen, with little reliance on laboratory investigations. The concept of accumulative micro-mercurial neurotoxicity with specific reference to dental amalgam, has been well documented and prolonged exposure to mercury has been associated with the unique lesions of the AD brain.

Therefore, amalgam, as the largest source of mercury vapor in the general population, should be included in the differential diagnosis of patients being investigated for neuro-psychiatric problems and shortterm memory loss.

Apo-E genotyping, a comparatively non-invasive, simple and relatively cheap laboratory test, warrants further investigation as an in vivo biomarker that could support the clinical differential diagnosis in the primary health environment, and help identify those symptomatic patients who are at greater risk of mercury toxicity and of AD.

Further supportive evidence of exposure and accumulative retention of mercury, can be obtained from a DMPS-provoked urine mercury challenge.