Airborne Aluminum as an Underestimated Source of Human Exposure: Quantification of Aluminum in 24 Human Tissue Types Reveals High Aluminum Concentrations in Lung and Hilar Lymph Node Tissues

Aluminum (Al) is the most abundant metal in the earth’s crust, and humans are exposed to Al through sources like food, cosmetics, and medication. So far, no comprehensive data on the Al distribution between and within human tissues were reported. We measured Al concentrations in 24 different tissue types of 8 autopsied patients using ICP–MS/MS (inductively coupled plasma–tandem mass spectrometry) under cleanroom conditions and found surprisingly high concentrations in both the upper and inferior lobes of the lung and hilar lymph nodes. Al/Si ratios in lung and hilar lymph node samples of 12 additional patients were similar to the ratios reported in urban fine dust. Histological analyses using lumogallion staining showed Al in lung erythrocytes and macrophages, indicating the uptake of airborne Al in the bloodstream. Furthermore, Al was continuously found in PM2.5 and PM10 fine dust particles over 7 years in Upper Austria, Austria. According to our findings, air pollution needs to be reconsidered as a major Al source for humans and the environment.


Table of Contents
Text S1.Chemicals and reagents, sample preparation and ICP-MS/MS measurements of patients 1-8 Table S1.ICP-MS parameters for tissue Al measurements Text S2.Chemicals and reagents, sample preparation and ICP-SFMS measurement of patients 9-20 Table S2.ICP-SFMS parameters Table S3.Patient information Table S4.Organ weights Table S5.Statistical information Table S6.Tissue water content Table S7.Total Al tissue content Samples of patients 1-8 were divided into three sets, every set was measured on a different day.No bone sample was provided for patient 3, and toenail instead of fingernail was provided for patient 6.
To validate trueness, the certified reference material BCR-639 (Joint Research Centre, Institute for Reference Materials and Measurements, European Commission, Geel, Belgium) was used for reference extractions.Furthermore, In and Re were used as internal standards during IP-MS/MS measurements.
Both were used as single element standards with a concentration of 1000 +/-3 mg • L -1-and were purchased from LabKings (Hilversum, the Netherlands).The quality control 26-element standard solution for calibration was purchased from LabKings (Hilversum, the Netherlands).
Sample preparation was performed in a cleanroom of ISO class 8, ICP-MS/MS measurements were performed in an ISO class 7 clean room.

Sample preparation for elemental analysis
After delivery to the laboratory, 5 mL of 30% HNO3 were added to each frozen (-18 °C) sample and transferred to PFA extraction tubes.Two BCR-639 plasma reference material and four blank extractions only containing HNO3 were included in every extraction run.During the first three extraction days, 250 µL of reference material were weighed and used for reference material extraction.Measurements were close to or below LLOQ, thus the volume was increased to 500 µL for all following reference material extractions.In total, 16 extractions with 500 µL, which were weighed before digestion, were performed yielding a median Al concentration of 214 +/-12 µg/L (median +/-standard deviation), which is in the certified range of 194 +/-14 µg/L.Due to high instrumentation cleaning efforts required after ICP-MS measurement of patients 1-5, 1 mL of 30% H2O2 was added to all extraction tubes of patients 6-8 to ensure full digestion of samples.Anton Paar Multiwave PRO was used for microwave digestion, where temperature increased to 180°C within 20 minutes, was constant at 180°C for 10 minutes followed by cooling to 70°C.Extracts were transferred into metal free tubes, diluted with 5 mL ultrapure water or, for samples containing 1 mL H2O2, 4 mL ultrapure water, and weighed.Samples were diluted 1 to 5 with ultrapure water by weighing, to reach a total HNO3 concentration of approximately 3%.Extracts with Al concentrations exceeding 1000 ppb were diluted 1 to 10 with 3% HNO3.After each sample digestion cycle, one cleaning cycle using HNO3 and applying the microwave program as for the sample digestion was performed to minimize the risk for carryover.
Quantification was performed by a 9-point matrix-matched external calibration with standard solutions containing Al ranging from 0.1 µg • L -1 -200 µg • L -1 .To validate trueness, certified reference material TM 35.2 (trace element matrix reference material made from filtered and diluted Lake Ontario water, certified for Al) was measured with every sample set.
The plasma reference material BCR-639 provides certified values for Al, Se and Zn.The sample preparation procedure and ICP-MS analysis were also suitable for the analysis of Se and Zn, which were thus also quantified.The clear focus of this study is Al, therefore the results of Se and Zn measurements were not provided in this publication.

ICP-MS/MS measurements
Inductively coupled plasma mass spectrometry (ICP-MS/MS) measurements were performed using Agilent 8800 ICP-MS/MS (Agilent Technologies, Tokyo, Japan) equipped with an CETAC ASX-520 autosampler (CETAC Technologies, Omaha, USA) and a MicroMist nebulizer operated at a sample
HNO3 was sub-boiled using a Savillex DST-1000 acid purification-system.Certified ICP single element standards for trace analysis were purchased from Inorganic Ventures (Al, Si and In at concentration of 1000 mg L -1 ).All laboratory consumables used for sample preparation and analysis were made of plastic material and double acid cleaned (HNO3/HCl, w = 10 %/2 % (v/v) and w = 1 %/0.2 % (v/v) 24 h each,

Sample preparation for elemental analysis
After delivery to the laboratory, the frozen (-18 °C) tissue samples were mineralized by microwave assisted acid digestion.Approximately 0.300 g of sample were accurately weighted into PTFE vessels of the microwave (Anton Paar, Graz, Austria, Multiwave 3000, rotor 16 HF 100).In a first step, sample digestion was performed with 4 mL of concentrated sub-boiled HNO3, 1 mL of H2O2 and 50 µL of HF at 1400 W for 15 min (+ 15 min ramp).In a second step, complexation of HF with H3BO3 was performed.Therefore, the microwave vessels were quickly opened after cooling down to approx.30 °C, 500 µL of super-saturated re-crystallized H3BO3 were added, vessels were closed again and another digestion cycle was performed at 1000 W for 10 min (+ 15 min ramp).After cooling down the vessels to room temperature, the obtained digests were quantitatively transferred into 50 mL mL PP-bottles and gravimetrically diluted to 30 g with sub-boiled water.After each sample digestion cycle, two cleaning cycles, using HNO3/H2O2/HF and applying the microwave program as for the sample digestions were performed to minimize the risk of analyte carry-over.
For analysis, the samples were, depending on the concentrations of the analytes, not diluted and further gravimetrically diluted by a factor of 5, 50 and 100, respectively, to fit within the applied working range (0.1 -100 µg L -1 ) and spiked with the internal standard to obtain a final concentration of 1 µg L -1 In in the sample.To minimize matrix induced measurement-and quantification errors, dilutions were performed using diluted digestion blanks and keeping the acid concentrations constant.
Quantification was performed by an 8-point matrix-matched external calibration with standard solutions of Al and Si ranging from 0.1 µg L -1 -100 µg L -1 and internal standardization via In.The CRM TM 35 was used for verification of the analytical method.To verify matrix induced signal intensities and resulting slopes, calibration standards were prepared in synthetic solutions containing 13.3 % (v/m) HNO3 + 0.17 % (v/m) HF, and solutions obtained by dilutions of digestion blanks and digested BOVM-1 with 13.3 % (v/m) HNO3 + 0.17 % (v/m) HF, respectively, by a factor of 10 and 100.

ICP-SFMS measurements
Quantification was performed on the ICP-SFMS Element2 High Resolution ICP-SFMS (ThermoFisher, Bremen, Germany) in the high-resolution mode (HR, m/Δm >10000), equipped with a PFA scott-type spray chamber and an injector made of sapphire (both AHF Analysentechnik).Measurements were performed by continuous acquisition (sample intake via PFA-ST nebulizer and a 0.2 mm i.d.uptake capillary (Elemental Scientific Inc,.ESI, Omaha) in self-aspiration mode (sample uptake:50 µL min -1 ).
Tuning parameters were optimized to obtain best possible sensitivity (min 1x10 6 cps/1 µg L -1 In in LR), signal stability and an oxide rate 238 U 16 O + / 238 U + ratio <5%.Instrumental parameters and masses monitored are shown in Table S2.

Figure S1 .
Figure S1.Lumogallion and H&E staining of patient 2 Figure S2.Quarterly averages of Al in PM10 between 2014-2020 Supplementary References rinsed three times with Milli-Q®-water and dried in a clean bench before single use.50 mL PE bottles used for sample storage were conditioned with 10 % (v/v) sub-boiled HNO3/0.2 % (v/v) HF after the acid cleaning procedure until further use.To validate trueness, certified reference materials TM 35 (trace element matrix reference material made from filtered and diluted Lake Ontario water, certified for Al) and BOVM-1 Bovine Muscle Certified Reference Material for Trace Metals and other Constituents (National Research Council Canada, information value for Al given), have been processed.Additionally, spike recovery experiments at two different concentration levels were conducted.Preparatory laboratory work for elemental analysis and measurements were performed in an ISO class 8 and an ISO class 7 clean room with laminar flow hoods ISO class 5 according to ISO 14644-1, respectively.

Table S1 . ICP-MS parameters for tissue Al measurementsTable S1 .
Instrumental parameters for ICP-MS/MS measurements of tissue Al concentrations

Table S2 .
Instrumental parameters for ICP-SFMS measurements of Al and Si in tissues of patients9-  20.

Table S3 . Patient informationTable S3 .
Information on cause of death, pre-existing conditions and medications of patients 1-20.

Table S4 . Organ weightsTable S4 .
For all patients except patient 2, organ weights of heart, lungs, liver, spleen and kidneys were determined during autopsy.NA = not available

Table S5 . Statistical informationTable S5 .
Data was tested for statistical significance between all of the following tissue types: fingernail, abdominal skin, lung -right upper lobe, lung -right inferior lobe, hilar lymph node,

Table S7 . Total Al tissue contentTable S7 .
The average lung was found to have a ten-times higher absolute Al amount than liver, the tissue with the second highest absolute Al content.<LOQ = below limit of quantification; NA = not