Context.—

Clinical testing for Wilson disease (WD) is potentially challenging. Measuring the fraction of labile bound copper (LBC) to total copper may be a promising alternative diagnostic tool with better sensitivity and specificity than some current biomarker approaches. A dual filtration–based inductively coupled mass spectrometry (ICP-MS) assay to measure LBC in serum was developed.

Objective.—

To establish a reference interval for LBC and LBC to total copper (LBC fraction) in a healthy adult population, and to examine associations between total copper, LBC, and LBC fraction with age, sex, menopausal status, hormone replacement therapy, and supplement use.

Design.—

Serum samples were collected from healthy male (n = 110) and female (n = 104) patients between the ages of 19 and 80 years. Total copper and LBC were analyzed using ICP-MS. Results were used to calculate the LBC fraction. Reference intervals were calculated for the 2.5th and 97.5th percentiles for both LBC and LBC fraction.

Results.—

The reference intervals for LBC were determined to be 13 to 105 ng/mL and 12 to 107 ng/mL for female and male patients, respectively. The reference intervals for the LBC fraction were 1.0% to 8.1% and 1.2% to 10.5% for female and male patients, respectively. No significant associations were found regarding age, menopausal status, hormone replacement therapy, or vitamin and supplement use.

Conclusions.—

Sex-specific reference intervals have now been established for LBC and LBC fraction. These data in conjunction with further testing of WD populations can be used to assess the sensitivity and specificity of LBC fraction in screening, monitoring, and diagnosis.

Copper is an essential trace element, participating in many biochemical pathways throughout the human body. In the healthy adult, approximately 100 mg of copper is stored throughout the body, primarily in the liver. Most copper in plasma is bound to the enzyme ceruloplasmin (Cp), a copper-dependent multifunction oxidase enzyme.1,2  In healthy individuals, 70% to 95% of copper circulating in the bloodstream is tightly protein bound.1–5  The remaining copper fraction is often referred to as “free” copper; however, a more accurate description is “labile bound copper” (LBC) because it is not truly circulating freely but is loosely bound to various smaller proteins, including albumin, transcuprein, tetrapeptides, and other amino acids.1,4,5 

At a cellular level, copper acts as a cofactor in many enzymatic redox processes, including the construction of the extracellular matrix, the synthesis of neurotransmitters, nucleic acid repair,6,7  cellular respiration, and cell proliferation.8  Proper maintenance of copper balance via transporter proteins is essential in preventing atherosclerosis and hypertension, healing wounds,7  and in the maintenance of normal metabolic and neurological functions.1  Excess oxidation caused by high LBC concentration can lead to oxidative stress due to the formation of reactive oxygen species, which can cause cellular damage at elevated levels and are associated with a wide range of diseases.1,9–13 

Wilson disease (WD) is an autosomal recessive hereditary condition occurring in approximately 1 in 30 000 individuals.14–16  Additionally, it is estimated that 1 in 90 individuals are heterozygous, asymptomatic carriers.16,17  The disease is characterized by a defect in the ATP7B enzyme, a membrane transport protein integral to copper metabolism, leading to accumulation of copper in the liver and a low total serum copper concentration.8–10,14  Liver dysfunction is a hallmark of WD, presenting in myriad patterns.18,19  WD symptoms typically manifest before the age of 40 years.19 

Improving the detection of WD can mitigate the deleterious effects of this disorder. A 2007 retrospective analysis of WD cases found that the number one cause of death in WD is delayed diagnosis.20  Liver biopsy and genetic testing, the gold standard in WD diagnosis, can be potentially costly and invasive.18  High 24-hour urinary copper, low total serum copper, and low serum Cp index are possible indicators of WD; however, reported sensitivity and specificity of these tests vary.14,16,18  A meta-analysis investigating the diagnostic accuracy of WD biomarkers, conducted in 2021 by Salman et al,21  found a wide range in the reported sensitivity (65%–99%) and specificity (59%–100%) of Cp index, as well as the reported sensitivity (50%–80%) and specificity (76%–98%) of 24-hour copper urine. Because WD is associated with a loss of Cp copper binding function, the concentration of non–ceruloplasmin-bound copper (NCC) is an attractive target as a diagnostic aid.22  NCC has been historically estimated by the following formula: NCC = total serum copper (µg/dL) – [3.15 × ceruloplasmin (µg/dL)]. This calculated estimate has limitations because it is difficult to directly measure Cp, and it assumes that all available Cp is fully saturated with copper.22–24  As such, a clinical assay that can directly quantify the “free” and/or labile bound fraction of total copper in serum is needed. Clinical interest in LBC extends beyond WD; the relationship of low NCC with rheumatoid arthritis has been investigated25  and more recently, literature suggests that LBC could be elevated in patients with Alzheimer disease (AD).23,26 

Direct measurement of LBC divided by total copper (LBC fraction) is believed to be an improvement on estimates yielded from the NCC formula.22  LBC fraction is a tool with potentially high sensitivity and specificity for the detection of WD.15,17  Various methods for the direct analysis of LBC (also referred to as “exchangeable copper”) have been developed in the past. A liquid chromatography–based method was developed by Quarles et al27  which effectively separated LBC from protein-bound copper. The most commonly referenced approach is the use of individual filtration vials to isolate LBC from total copper.24,28,29  At present, reference intervals found in the literature17,24  are limited by sample size,17,24  age of participants,30  and a lack of metal-free collection tubes.29,30 

The aim of this study was to develop a high-throughput inductively coupled plasma mass spectrometry (ICP-MS)–based method for the accurate determination of LBC in human serum collected in trace metal–free tubes via simultaneous 96-well plate filtration, to aid in the assessment of copper related disorders. By measuring both LBC (ng/mL) and total copper concentration (ng/mL) in a healthy population, a reference interval for both LBC and the calculated LBC fraction in a healthy adult population can be established as a baseline metric for use in the evaluation of copper-related disease abnormality.

LBC is isolated through a serially centrifugation-based filtration process beginning by removing high–molecular weight (high-MW) copper-binding proteins from serum. The remaining low-MW proteins loosely bind the remaining copper. This loosely bound copper is removed through EDTA chelation31  and filtered to isolate the total LBC fraction; it is then quantified via ICP-MS (Figure 1).

Figure 1.

Labile bound copper is isolated from total copper through a series of steps using molecular weight cutoff (MWCO) filters and ethylene diamine tetraacetic acid (EDTA) chelation. A, High-molecular-weight proteins, including ceruloplasmin and transcuprein, bound to copper are filtered out of serum using a 100-kDa MWCO filter. The filtrate is then (B) incubated at 37°C for 1 hour to chelate the remaining copper, including that bound to albumin, to EDTA. Finally, the serum is (C) filtered through a 30-kDa MWCO filter, removing most of the remaining proteins and leaving the labile bound copper fraction in the filtrate.

Figure 1.

Labile bound copper is isolated from total copper through a series of steps using molecular weight cutoff (MWCO) filters and ethylene diamine tetraacetic acid (EDTA) chelation. A, High-molecular-weight proteins, including ceruloplasmin and transcuprein, bound to copper are filtered out of serum using a 100-kDa MWCO filter. The filtrate is then (B) incubated at 37°C for 1 hour to chelate the remaining copper, including that bound to albumin, to EDTA. Finally, the serum is (C) filtered through a 30-kDa MWCO filter, removing most of the remaining proteins and leaving the labile bound copper fraction in the filtrate.

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Reagents

All reagent preparation, sample preparation, and sample analysis for this study took place at the Mayo Clinic Metals Laboratory (Rochester, Minnesota), an ISO class 7 cleanroom. Reagents in this study were prepared using MilliQ deionized water, referred to as special reagent water (SRW). Reagents used in sample preparation include 1% Ultra-Trace Optima grade HNO3 (Fisher Scientific, Pittsburgh, Pennsylvania), pH 7.4 phosphate-buffered saline (PBS; Millipore Sigma, St Louis, Missouri), 0.5 M UltraPure ethylene diamine tetraacetic acid (EDTA; Fisher Scientific), and 10 mM histidine stock solution (Millipore Sigma). Diluent was prepared with a concentration of 0.8 mM EDTA (Fisher Scientific), 5 µg/L lithium (Inorganic Ventures, Paterson, New Jersey), 2% NH4OH (v/v) (GFS Chemicals, Columbus, Ohio), and trace NaOH (Fisher Scientific), referred to as LBC Diluent. An internal standard inline solution was made with 0.1% Triton X-100 (Fisher Scientific) and contained 1200 ng/mL gallium and 60 ng/mL rhodium (both Inorganic Ventures), 10% t-butanol (Acros Organics, Geel, Germany), and 2% HCl (Millipore Sigma). Three levels of quality control (QC) samples were prepared in house. For the low QC, bovine calf serum was used (Cytiva Life Sciences, Marlborough, Massachusetts). The medium-level QC consisted of 1% bovine serum albumin in PBS, and a 200 ng/mL copper standard (Inorganic Ventures) was used for high QC. All method validation studies were completed using either leftover de-identified human serum samples or National Institute of Standards and Technology (NIST) standard reference materials (SRM) 1640 and 1643e (National Institute of Standards and Technology, Gaithersburg, Maryland).

Isolation of LBC

MW size exclusion 96-well PALL filter plates (Pall Corporation, Port Washington, New York) were used to separate the labile copper fraction from total copper in serum. Prior to serum loading, 100-kDa MW filter plates were placed on a 96-well deep-well plate and washed with 200 µL of 1% HNO3 per well, then centrifuged for 5 minutes at 2113g using a Hettich Rotanta 460 centrifuge equipped with a swing-bucket rotor with a radius of 21 cm (Hettich, Tuttlingen, Germany). The HNO3 was then discarded, and the process was repeated using SRW. The washed filter plates were loaded with 120 µL of PBS followed by 30 µL of sample, then centrifuged for 2 hours at 3756g. Thirty-kDa MW filter plates were then prepared for use following the same wash procedure as the 100-kDa plates. A chelation solution was prepared with 45.5 mL of 0.5 M EDTA and 22.8 mL of 10 mM histidine stock solution, filled to 0.5 L with SRW. Using an Integra Viaflo 96 Channel Electronic Pipette (Integra Biosciences, Hudson, New Hampshire), 75 µL of chelation solution followed by 75 µL of filtrate from the 100-kDa MW well plate was added to the 30-kDa MW filter plate. The plate was then incubated using a Revsci Incufridge 233 Basic (Revolutionary Science, Shafer, Minnesota) at 37°C for 1 hour to catalyze the chelation reaction. After the incubation period, the plate was centrifuged for 1 hour at 3756g. The filtrate was then ready for sample analysis.

Sample Analysis

Analysis of LBC was performed using a Perkin Elmer NexION 2000 ICP-MS (Perkin Elmer, Waltham, Massachusetts) to directly measure the copper (65Cu) concentration. The instrument was run in kinetic energy discrimination mode using helium as a nonreactive gas. See Table 1 for method parameter details. A sample volume of 50 µL of serum filtrate was diluted with 950 µL of LBC diluent into a Greiner 96-well deep well plate. A 6-point calibration of the analytic measurement range (AMR) was performed daily prior to sample analysis using Inorganic Ventures standards with concentrations of 0, 1, 10, 50, 100, and 200 ng/mL copper (Inorganic Ventures). Two levels of calibration-specific QC samples were run with the calibration, 1 of which was prepared in house from an acidified urine pool, the other of which was obtained from UTAK (UTAK Laboratories Inc, Valencia, California). In-house QC samples were included in each run to monitor assay performance, undergoing the same filtration and dilution process as the rest of the samples. PBS blanks were included at the beginning of each filter plate to assess cleanliness. All samples were analyzed in triplicate.

Table 1.

Inductively Coupled Plasma Mass Spectrometer Operating Conditions

Inductively Coupled Plasma Mass Spectrometer Operating Conditions
Inductively Coupled Plasma Mass Spectrometer Operating Conditions

Total copper analysis was performed on a Perkin Elmer NexION 350 ICP-MS. As with LBC, the 65Cu isotope was measured directly. The instrument was run in dynamic reaction cell mode, with ammonia (NH3) as the reaction gas. See Table 1 for method parameter details. Sample volumes of 100 µL of serum were diluted with 2400 µL of EDTA diluent (10 ng/mL 6Li in 10 μM EDTA and trace NaOH) into 13 × 75 Sarstedt polystyrene test tubes (Sarstedt, Nümbrecht, Germany). A 6-point calibration was performed daily using Inorganic Ventures standards with concentrations of 0, 10, 50, 100, 250, and 500 μg/dL copper (Inorganic Ventures). Three levels of UTAK QC materials were used throughout analysis (UTAK Laboratories). This method is a clinically validated laboratory developed test that is used for routine analysis of clinical serum samples.

Method Validation

The analytic method was tested and validated following Clinical and Laboratory Standards Institute (CLSI) EP guidelines. Precision was evaluated in 2 ways: intra-assay and inter-assay. To test intra-assay precision, 6 serum samples spanning the LBC AMR were evaluated 20 times within 1 run. Inter-assay precision was tested by evaluating 6 serum samples spanning the AMR, across 20 different runs. The acceptance criteria were set to pass with a coefficient of variance (%CV) of 40% or less for samples with LBC concentrations between the lower limit of quantification (LLOQ) and 100 ng/mL, and 20% or less CV for samples more than 100 ng/mL.32  The limit of blank (LOB), limit of detection (LOD), and LLOQ were all determined to evaluate the assay sensitivity. Sixty replicates of PBS as blank material and 60 replicates of a serum sample with an LBC concentration of approximately 15 ng/mL were analyzed and used to determine the LOB (|meanBlank| + 1.645 * SDBlank) and the LOD (LOB + 1.645 * SDSample). The LLOQ was determined by using the precision data (Table 2) to extrapolate the concentration where inter-assay precision CV was 40% or less.33  Recovery and linearity experiments were used to evaluate the accuracy of the assay. Recovery was assessed by spiking samples of PBS, NIST SRM 1640, and NIST SRM 1643 with a 1:1 ratio of LBC standards (0, 10, 100, and 200 ng/mL). All spiking was done prior to the LBC isolation preparation process. Acceptable recovery limits were neat and spiked sample results matching within the greater of ±25% or 20 ng/mL.34  Linearity was first evaluated using 5 aqueous samples (NIST SRMs and Inorganic Ventures Cu standards) with LBC concentrations of 22, 93, 200, 500, and 1000 ng/mL analyzed during 5 runs. The samples were evaluated neat, diluted ×2 with PBS, and diluted ×10 with PBS. This experiment was conducted in 2 ways. In the first, the samples were diluted prior to any MW filtration. In the other, the dilution took place using the resulting filtrate from the LBC isolation process. Linearity was evaluated using 5 serum samples with endogenous LBC concentrations of 19, 94, 336, 406, and 729 ng/mL analyzed during 5 runs. The samples were evaluated neat, diluted ×2 with PBS, and diluted ×10 with PBS prior to MW filtration. In all experiments the acceptance criteria were that diluted results match expected results, calculated from the neat sample, within the greater of ±10% or 20 ng/mL.

Table 2.

Mean Labile Bound Copper (LBC) Precision Results Collected for 20 Replicates Within Run (Intra-Assay) and Between Runs (Inter-Assay)

Mean Labile Bound Copper (LBC) Precision Results Collected for 20 Replicates Within Run (Intra-Assay) and Between Runs (Inter-Assay)
Mean Labile Bound Copper (LBC) Precision Results Collected for 20 Replicates Within Run (Intra-Assay) and Between Runs (Inter-Assay)

Reference Interval Sample Collection

Eligible healthy adult serum donors were recruited and consented by Mayo Clinic Quality Management Services from an existing biospecimen donor pool. Screening was completed by Quality Management Services prior to recruitment to exclude donors with the following diagnoses: WD, acute hepatitis, chronic hepatitis, renal failure, biliary cirrhosis, celiac disease, and leukemia, among other renal, biliary, digestive, and autoimmune disorders. Questionnaires were used to collect data from each participant about possible metal exposures through metal implants, vitamin and mineral supplements, seafood, industrial welding or smelting, kidney disease, hormone replacement therapy, and contrast agents given with computed tomography or magnetic resonance imaging scans. Retrospective medical chart review was completed by the Mayo Clinic Department of Quantitative Health Sciences, Division of Clinical Trials and Biostatistics, to ensure that donors did not meet any of the exclusion criteria, determine menopausal status, and identify donors using hormone replacement medication. Donors were asked to avoid taking zinc-containing vitamins or antiviral drugs prior to sample collection. It was determined by the Mayo Clinical Institutional Review Board that this study was classified as a Quality Improvement project, and in accordance with the Code of Federal Regulations, 45 CFR 46.102, did not require Institutional Review Board review.

All samples were de-identified upon collection to protect patient confidentiality. Whole blood was collected in nonadditive BD Vacutainer Trace Element Tubes (BD, Franklin Lakes, New Jersey). Whole blood samples were centrifuged at 2113g for 10 minutes. The resulting serum was immediately poured off into a trace metal–free tube and stored at −80°C for at least 48 hours prior to analysis. LBC and total copper results were collected for each sample, following the procedures outlined above.

Statistical Analysis

Nonparametric Kolmogorov-Smirnov tests were used to compare the distribution of total copper, LBC, and LBC fraction results by sex, menopausal status, and questionnaire responses to taking hormone replacement and to taking a vitamin or supplement. Using guidelines from CLSI document EP28-A3c to verify a reference interval, it was assessed whether 10% or fewer samples were outside the current total copper reference interval.35  Nonparametric quantile regression was used to determine the relationship of the mid-95th percentiles for total copper, LBC, and LBC fraction partitioned for sex and age. Spearman rank correlation was used to determine the measure of association between total copper, LBC, and LBC fraction with increasing age. Quantile regression CIs and P values were calculated using a bootstrap resampling procedure with 10 000 replicates. It was assessed whether more than 4% of female or more than 4% of male patients were below the lower limit of the overall reference interval (or above the upper limit) to suggest a clinically significant difference between sex per CLSI document EP28-A3.35  Analyses were performed using SAS version 9.4 (SAS Institute Inc, Cary, North Carolina). P < .05 was considered statistically significant.

Method Validation

The intra-assay and inter-assay precision results are summarized in Table 2. All results were deemed acceptable by predetermined acceptance criteria. The highly manual sample preparation process and instrument LOD both contribute to the high levels of imprecision seen at low LBC values. The instrument LOB and LOD were determined to be 0.3 ng/mL and 0.7 ng/mL, respectively (to convert to micromoles per liter, multiply by 0.016). When the 10-fold dilution of the sample preparation process is accounted for, this corresponds to an assay LOB, LOD, and LLOQ of 3, 7, and 19 ng/mL, respectively. Recovery was calculated as (measured result/expected result) × 100%. The recovery samples went through the entire preparation and analysis process. The recovery of copper standards with concentrations of 10, 100, and 200 ng/mL was found to be 82%, 96%, and 99%, respectively, in PBS and 95%, 109%, and 99%, respectively, in NIST water specimens.

Linearity experiments showed accuracy across the AMR, with an instrument upper limit of quantification determined to be 200 ng/mL, allowing measurement of samples with up to 2000 ng/mL LBC when considering the ×10 dilution factor in the sample preparation. Linear response throughout the AMR was calculated as (measured result/expected result) × 100%. Aqueous samples that were diluted ×2 and ×10 with PBS after the LBC isolation process had average recoveries of 96%, 94%, 98%, 99%, and 100% at neat concentrations of 22, 93, 200, 500, and 1000 ng/mL. At the same concentrations, samples that were diluted prior to the full LBC isolation process yielded an average of 88%, 97%, 89%, 100%, and 95% recovery. Serum samples that were diluted ×2 and ×10 prior to the full LBC isolation process yielded an average recovery of 100%, 87%, 97%, 92%, and 92% at neat concentrations of 19, 94, 336, 406, and 729 ng/mL (Figure 2).

Figure 2.

Recovery of labile bound copper across the analytic measurement range for (A) aqueous samples diluted after the filtration process, (B) aqueous samples diluted before the filtration process, and (C) serum samples diluted before the filtration process.

Figure 2.

Recovery of labile bound copper across the analytic measurement range for (A) aqueous samples diluted after the filtration process, (B) aqueous samples diluted before the filtration process, and (C) serum samples diluted before the filtration process.

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Stability

Experiments were conducted to measure the stability of LBC at room temperature, refrigerated (4°C), and at 2 frozen temperatures (−20°C and −80°C) for serum samples collected from healthy individuals (n = 12). Because of a suspected dilution error affecting all samples on day 1 of analysis, day 7 was used as a baseline. After 29 days, the average percent change from baseline concentration was +60% (room temperature), +34% (4°C), −9% (−20°C), and −12% (−80°C). A second study was conducted to analyze stability between days 0 and 8 (n = 8) at room temperature, 4°C, and −80°C. The average percent change from baseline concentration was +27% (room temperature), +20% (4°C), and +20% (−80°C); samples were therefore determined to be stable for up to 29 days when stored at −20°C or −80°C. These results align with stability experiments previously documented in the literature.17,23  An additional experiment showed that the serum filtrate from the filtration preparation process could be refrigerated at 4°C for 24 hours prior to analysis, with an average percent difference of +1% in concentration before and after refrigeration.

Reference Interval

Serum was collected from a total of 214 patients between the ages of 19 and 80 years; 110 men and 104 women. Total copper, LBC, and LBC fraction values were obtained for all samples. In general, total copper was higher in women (median, 1145 ng/mL) than in men (median, 923 ng/mL; P < .001), and LBC fraction was lower in women (median, 2.6%) than in men (median, 3.4%; P = .003). No statistically significant differences were found when total copper, LBC, and LBC fraction distributions were compared by menopausal status, hormone replacement therapy, or vitamin and supplement intake (all P ≥ .14; Table 3).

Table 3.

Distributions by Sex, Menopausal Status, and Questionnaire Responses

Distributions by Sex, Menopausal Status, and Questionnaire Responses
Distributions by Sex, Menopausal Status, and Questionnaire Responses

The sex-specific total copper reference interval currently used in the Mayo Clinic Metals Laboratory of 770 to 2060 ng/mL for female and 730 to 1290 for male individuals was verified by this data set; only 7 women (6.7%) and 11 men (10.0%) were outside the respective reference intervals (Figure 3).36  The experimentally derived sex-specific LBC reference intervals were found to be 13 to 105 ng/mL (median, 29 ng/mL) and 12 to 107 ng/mL (median, 31 ng/mL) for women and men, respectively (Figure 4, A; Table 4). The sex-specific LBC fraction reference intervals were found to be 1.0% to 8.1% and 1.2% to 10.5% for women and men, respectively (Figure 4, B; Table 4). The 97.5th percentile of total copper was higher in women than in men (P < .001); no other significant relationships within the mid-95th percentiles were found between LBC or LBC fraction with age or sex (all P ≥ .07; data not shown). A statistically significant positive correlation was found between total copper and age (rs = 0.15; P = .02), and a negative correlation was found between both LBC (rs = −0.15; P = .03) and LBC fraction (rs = −0.18; P = .009) with age. When stratified by sex, significant correlations were found between increasing total copper with increasing age for men (rs = 0.21; P = .03) and decreasing LBC fraction with increasing age for men (rs = −0.22; P = .02).

Figure 3.

Verification of sex-specific total copper reference interval for (A) women and (B) men. The blue and pink lines indicate the 2.5th and 97.5th percentiles.

Figure 4. Overall and sex-specific reference intervals for (A) labile bound copper (ng/mL) and (B) labile bound copper fraction (%). The black lines are estimates of overall reference interval, the pink lines are estimates of the female reference interval, and the blue lines are estimates of the male reference interval. All reference intervals are the mid-95th (2.5%–97.5%) percentiles.

Figure 3.

Verification of sex-specific total copper reference interval for (A) women and (B) men. The blue and pink lines indicate the 2.5th and 97.5th percentiles.

Figure 4. Overall and sex-specific reference intervals for (A) labile bound copper (ng/mL) and (B) labile bound copper fraction (%). The black lines are estimates of overall reference interval, the pink lines are estimates of the female reference interval, and the blue lines are estimates of the male reference interval. All reference intervals are the mid-95th (2.5%–97.5%) percentiles.

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Table 4.

Overall and Sex-Specific Reference Intervals

Overall and Sex-Specific Reference Intervals
Overall and Sex-Specific Reference Intervals

Using 100- and 30-kDa MW cutoff filters in conjunction with 96-well plates, a robust assay has been created that allows for the direct measurement of LBC in human serum. One 96-well filter plate can hold up to 32 patient and quality control samples run in triplicate, making this an easily scalable and affordable assay to offer clinically. The AMR of the test allows for the direct quantification of samples with LBC concentrations from 19 to 2000 ng/mL, which is sufficient to assess the upper and lower ends of the reference interval. Sample stability (29 days frozen at −80°C) was consistent with what has been previously reported by El Balkhi et al.28  Recovery and linearity experiments confirmed the accuracy of the assay. It is important to note that for recovery and linearity experiments, samples were diluted and/or spiked prior to the preanalytic preparation process, thus measuring the performance of the entire LBC method, including the filtration process, and not just the ICP-MS portion of the method.

The overall reference interval established in these experiments for LBC (12–105 ng/mL) is comparable to adult reference intervals previously reported for filtration-based methodologies by El Balkhi et al28  (36–71 ng/mL) and McMillin et al30  (0–102 ng/mL). The mid-95th percentile non–sex-specific interval that we established is most like the pediatric reference interval (ages 1–18 years) reported by Yim et al29  (26–101 ng/mL). Total copper results obtained in this reference interval study were used to confirm the previously internally established sex-specific reference intervals for total serum copper in adult male and female individuals (730–1290 ng/mL and 770–2060 ng/mL, respectively).36  It has been observed that female and male individuals differ regarding total copper serum reference interval concentrations. This has been partially attributed to the ability of oral contraceptives to elevate serum total copper concentrations.37  The overall reference interval for LBC fraction (1.1%–9.6%) determined in this study is also similar to those reported by El Balkhi et al17  (3.2%–8.6%) and Yim et al29  (3.2%–10.5%). It is important to note that 4.8% of women had LBC fraction values below the overall reference interval, suggesting the clinical significance of sex-specific LBC fraction intervals. The lower LBC fraction reference values in women compared with men may be due to higher total serum copper reference interval values in women.

In addition to evaluating LBC (ng/mL), total copper (ng/mL), and LBC fraction intervals based on sex, there was interest in determining if menopausal status, increasing age, hormone replacement data, and vitamin and supplement use would affect the LBC, total copper, or LBC fraction in the study population. As is shown in Table 3, no statistically significant associations were found between menopause, hormone therapy, vitamin or supplement use, and total copper, LBC, or LBC fraction. Data on contraceptive use were not collected, so the effects of hormonal birth control on LBC were not assessed. Furthermore, the small numbers of individuals in this study who were on hormone replacement may preclude conclusions regarding the effects of hormone replacement status. As shown in Table 5, a slight increase of total copper and decrease of LBC and LBC fraction were seen with age. It should be noted when interpreting these results that the sample population is skewed toward younger individuals (ages 19–40 years, n = 110; ages 41–60 years, n = 73; ages 61–80 years, n = 31).

Table 5.

Spearman Correlation of Total Copper, Labile Bound Copper, and Labile Bound Copper Fraction With Age

Spearman Correlation of Total Copper, Labile Bound Copper, and Labile Bound Copper Fraction With Age
Spearman Correlation of Total Copper, Labile Bound Copper, and Labile Bound Copper Fraction With Age

Because untreated WD is typically characterized by both a low total serum copper and a high LBC concentration,14  the LBC fraction potentially holds clinical utility because it accounts for both factors. Because of the difficulty of obtaining samples from untreated WD patients, this research relied on literature to review LBC fraction values in untreated WD patients. A study conducted by El Balkhi et al17  in 2011 analyzed total serum copper, total Cp, LBC, LBC fraction, urinary copper, and NCC calculation in 16 patients with WD, confirmed via genetic testing. All 16 patients had LBC fraction values between 23% and 86%, and the cutoff for WD diagnosis was set at greater than 18.5%. Of all diagnostic methods tested, LBC fraction was the only method that had no false-positive or false-negative WD diagnoses.17  A study published by Trocello et al38  in 2014 showed similarly promising results. A total of 21 patients were analyzed: 5 with confirmed WD, 14 heterozygous ATP7B carriers, and 2 controls. All 5 WD patients had LBC fraction values between 18% and 58%, whereas the highest result in a non-WD patient was 13.8%.38  The separation between the high end of our LBC fraction reference interval (8.1% for women and 10.5% for men) and the reported low end of LBC fraction in previously reported WD samples is promising.

Although LBC has been shown to be a promising diagnostic tool for WD, it may be applicable to other diseases as well. It is known that LBC can easily cross the blood-brain barrier; the brain is the organ with the second highest copper concentration behind the liver.1  Recent studies have investigated the role of LBC in the progression of AD. Copper, zinc, and iron have all been implicated in the “metal hypothesis” of AD, which postulates that the high levels of reactive oxygen species seen in AD are caused in part by a dysregulation of redox pathways, leading to excess oxidation by these metals.39,40  Direct redox reactions of LBC with the well-established AD biomarker amyloid-β add to this oxidative stress, and copper promotes both the dimerization of amyloid precursor protein and the production of Aβ itself.41  Additionally, inactive ceruloplasmin has been detected in the serum of patients with AD, resulting in an increase of LBC due to reduced Cp binding function.4  Finally, a genetic link has been found between various ATP7B gene variants with the risk of AD.1,26  Further work is needed to investigate the role that copper plays in the progression of AD, but the groundwork has been laid for the potential usefulness of LBC in learning about AD.

This study does have some limitations. A reference interval was determined for LBC and LBC fraction in healthy adults, but further work is needed to determine the specificity and sensitivity of this assay for WD diagnosis. A single study conducted by Guillaud et al15  in 2018 found that the LBC fraction results of WD patients (N = 9) were significantly higher than patients with non-Wilsonian hepatic diseases (N = 103). Further studies will be needed to confirm the diagnostic utility of this assay for WD. Additionally, this study is limited by the LLOQ of the assay. Although the AMR covers the normal reference interval of LBC concentrations, we currently do not have the precision to accurately assess potential labile copper deficiency. Currently, nearly all interest in LBC is related to an excess, not a deficiency.

This study has established a robust reference interval of LBC and LBC fraction in a healthy adult population. The robust analytic method used to conduct this study will soon be clinically available as a laboratory-developed test for health care providers to use in further investigating the role of LBC in WD, AD, and other disorders.

The authors would like to thank the Mayo Clinic Quality Management Services and Biospecimen Program Team for helping coordinate the collection of reference interval samples, the Mayo Clinic Department of Quantitative Health Sciences for providing statistical analysis of the reference interval data set, and Alexion Pharmaceuticals for providing funding for this research.

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Author notes

Financial support for this study was provided by Alexion Pharmaceuticals. The funders of this study had no role in its design, or the collection, analysis, and interpretation of data.

The authors have no relevant financial interest in the products or companies described in this article.