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Differentiation Between Nutritional and Carcinogenic Selenium via Speciation AnalysesRussell GeradsBusiness Development DirectorBrooks Applied Labs

Presented at the AOAC Conference, September 29, 2015

Presentation Outline:

I thought selenium was a nutrient…Selenium sourcesApplied Analytical MethodsAnalytical FindingsImplications for Humans, Cattle, Fish, and the Ecosystem

I Thought Selenium was a Nutrient…

Selenium is an essential trace element

Deficiency is widespread and causes increased cancer ratesEfforts to enrich nutrition in Se are underway in many areas

Reduces certain types of cancer (antioxidant in the form of glutathione peroxidase)

Reduces As toxicity (Se-As complexes)

Reduces Hg bioaccumulation (Se-Hg complexes)

I Thought Selenium was a Nutrient…

Se biomagnifies (review in a few minutes)

Effects observed:TeratogenicityMutagenDecreases reproductive efficiencyCirrhosis of the liver

Most organic-Se compounds are readily absorbed (increased uptake), but inorganic Se species dominate in the environment; somewhere along the process, conversion of inorganic Se into organic Se species must occur (rate limiting step)Selenate mobilized through sulfate chemical pathways for plants (greater efficiency than selenite)

I Thought Selenium was a Nutrient…

I Thought Selenium was a Nutrient…

I Thought Selenium was a Nutrient…

Biochemical Implications

Sulfur and oxygen analogues

Volatile methylated compounds(CH3-Se-CH3, CH3-Se-Se-CH3, CH3-SeO2-CH3)

Se-amino acids and Se proteinsSelenoamino acids (sulfur analogues)SelenomethionineSelenocysteineOther…

Biochemical Implications

Which Selenium is Nutritional?

Which Selenium is Nutritional?

Source: Rigby, M., Gerads, R., Lemly, D. Environmental Science Process & Impacts 2014

Selenium SourcesElectronics (diodes, photoelements, rectifiers, LEDs)Vulcanizing agent for rubberMedicine / nutritional supplementVegetation, groundwater, feed, food additives (human too!)Coal combustion (fly ash)Flue gas desulphurization (FGD)Oil refiningManufacturing gas plantsCoal and phosphorus miningRunoff from agricultural irrigation in arid regionsSe is produced from anodic sludge in copper refining andfrom lead chamber sludge in sulfuric acid production

Selenium Sources

Selenium production from copper mines and anode mud from electrolytic Cu refineries:

Cu2Se + Na2CO3 + 2O2 2CuO + Na2SeO3 + CO2

Acidified with sulfuric acid forming H2SeO3

H2SeO3 + 2SO2 + H2O Se(s) + 2H2SO4

Applied Analytical Methods

Size exclusion inductively coupled plasma dynamic reaction cell mass spectrometry (SEC-ICP-DRC-MS) – Selenoproteins and selenoaminoacids

Ion chromatography inductively coupled plasma dynamic reaction cell mass spectrometry (IC-ICP-DRC-MS) – Se(IV), Se(VI), SeCN, MeSe(IV)

Reverse phase chromatography inductively coupled plasma dynamic reaction cell mass spectrometry (RP-ICP-DRC-MS) – Seleno-amino acids and other small molecular metabolites

Extraction for selenoamino acids must be specific to intent: seleno-amino acids versus selenoproteins

Different selenoamino acids and proteins applied to inorganic selenium extraction method to confirm molecular degradation did not transpire

Applied Analytical Methods

Many published methods use or reference EXAFS/XANES

Problem:Utility of method limited to adjacent atoms

Many gross assumptions made regarding molecular forms using x-ray spectrometry

For more information please read:

Rigby, M., Lemly, D., Gerads, R. “Fish toxicity testing with selenomethionine spiked feed – what's the real question being asked?” Environmental Sci.: Processes & Impact. 2014, 16, 511.

Selenium Speciation Analysis by SEC-ICP-MS

Computer Waste

Column Oven

ICP-MS

AutosamplerLC Pump

A B

Column

Selenium Speciation Analysis by SEC-ICP-MS

Size exclusion inductively coupled plasma mass spectrometry (SEC-ICP-MS) – Selenoproteins and selenoamino acids

Flow

Large moleculesSmall molecules

Molecular identification by retention times for known standards (macro molecules require monitoring for other elements)

Selenium Speciation Analysis by SEC-ICP-MS

SeCN

Se-amino

Se(IV)

SeP

Selenium Speciation Analysis by IC-ICP-MS

Ion chromatography inductively coupled plasma dynamic reaction cell mass spectrometry (IC-ICP-DRC-MS) – Se(IV), Se(VI), SeCN, MeSe(IV)

Flow

Large moleculesSmall molecules separated by ionic interactions

Molecular identification by retention times for known standards

Selenium Speciation Analysis by IC-ICP-MS

Selenium Speciation Analysis by RP-ICP-MS

Reverse phase chromatography inductively coupled plasma mass spectrometry (RP-ICP-DRC-MS) – Se(IV), Se(VI), selenomethionine, selenocysteine, methylselenocystein, other seleno-amino acids

Flow

Large moleculesSmall molecules separated by carbon and ionic interactions

Molecular identification by retention times for known standards

Selenium Speciation Analysis by IC-ICP-MS

Full Time Range EIC(78) : 017SMPL_130827-ICPMS7-IC-SeMet-2_130827-ICPMS7-IC-SeMet Crunch.D

RT(min)

4.0 8.0 12.0Count

3x10

0

1

2

nd Name=O-setmet Exp. RT=2.89312 Det. RT=2.91960 Area=6407 Height=366

Compound Name=SetMet Exp. RT=5.69687 Det. RT=5.76248 Area=595259 Height=31104

Compound Name=Se4 Exp. RT=7.78530 Det. RT=7.78530 Area=11892 Height=1604

Enzymatically extracted yeast sample analyzed by RP-ICP-MS

>85% SeMe

Selenium Speciation Analysis by IC-ICP-MS

Full Time Range EIC(78) : 085SMPL_140414-SeMet.D

RT(min)

4.0 8.0 12.0

Count

2x10

0

2

4

Compound Name=SeMet Exp. RT=3.347 Det. RT=3.347 Area=49 Height=13

Compound Name=Se4 Exp. RT=4.328 Det. RT=4.047 Area=229947 Height=31053 Enzymatically extracted yeast sample analyzed by RP-ICP-MS

>90% Se(IV)

Implications for Humans, Cattle, Fish, and the Ecosystem

Toxicological risk dictated by organism

Not all food additives are the same “Selenized yeast”

Risk concerns: Human < Cattle < Fish < Birds

Selenium supplementation for cattle routine Application of incorrect molecular form of selenium may have negative effects on yield

Application of cattle waste to agriculture can have negative side effects depending on cattle feed

Increased attention from FWS, USDA, and EPA for agricultural runoff resulting in higher costs for industry over time

Acknowledgments

Thanks! to:

Brooks Applied Labs staff

Dr. Hakan Gurleyuk