Lead from Saliva

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Lead from Saliva

• Presented by Thomas McCormick, CEO
• Presentation compiled by Melissa Love and Shawn
  Maurer
• Original research conceived and developed by
• Dr. David Schneider, Chairman
• Assistance in sample collection and testing by
  Patriot Drug Testing
• Duane Fouts, R.Ph. and Erik Cissell
• Western Slope Laboratory, LLC
• and the North Carolina State Laboratory

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Agenda

• Present
• It is well-known that ingested lead is toxic,
  especially to children
• The current testing standard for lead uses whole
  blood
• CDC recognized limits of quantification of the
  whole blood test are 10 µg/dL
• Phlebotomists and special disposal infrastructure
  is necessary to test whole blood
• Future
• A test with a lower limit of quantification could
  help doctors and researchers better understand
  the effects of lead doses below 10 µg/dL on
  childrens development
• A saliva test would not require phlebotomists and
  special disposal infrastructure and would be
  preferred by children
• SolutionThe WSL Saliva Lead Test?
• Preliminary studies of the WSL Saliva Lead Test
  indicate that it holds promise as a screen
• Further studies may demonstrate a 11 correlation
  at doses above 10 µg/dL
• Potentially the WSL Saliva Lead Test can be shown
  to be accurate below 10 µg/dL as well

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Lead in the Body

• Although death from lead poisoning is rare, lead
  has many toxic effects
• Accumulation is gradual
• Elevated blood pressure occurs at levels less
  than 10 µg/dL
• This is the most common effect of lead toxicity
  in adults
• It contributes to the hypertension epidemic
• Headache, muscular weakness, and memory loss
  follow at higher doses
• At doses higher than 40 µg/dL, kidney problems
  and infertility result 1

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Lead Is Toxic for Children 1

• Children are at greater risk for lead toxicity
  than adults
• Childrens bodies absorb more ingested lead
• Children put hands and other objects in their
  mouths more frequently
• The fetal brain is especially at risk due to the
  immaturity of the blood-brain barrier
• Lead interferes with normal neurodevelopment

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Despite Risk Reduction, Threat Remains

• Extensive media coverage has raised awareness 2
• Risk has been greatly reduced over the last
  several decades via 1
• Removal of tetraethyl lead from gasoline
• Elimination of lead-solder from food cans
• Removal of lead from plumbing and paint
• Lead sources remain in the environment 3
• Lead-based paint
• Solder on pipes
• Some foreign candies
• Chinese toys
• Hobbies such as making stained-glass windows
• Occupational hazards

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At-Risk Populations 4,5

• Although legislation is in place to reduce
  exposure, approximately 310,000 US children ages
  one to five have blood levels above 10 µg/dL
• About 1.5 of the population
• Medicaid enrollees account for about 80 of US
  children ages one to five with whole blood lead
  levels greater than 20 µg/dL
• Programs such as the Childhood Lead Poisoning
  Prevention Program (CLPPP) help many states test
  for lead
• Some states require all Medicaid-enrolled
  children to be screened
• If screened levels are higher than the 10 µg/dL
  cutoff, follow-up testing and treatment may be
  necessary

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Neurodevelopmental Effects of Lead in Children

• 2-4 point drop in IQ with each 1 µg/dL in the
  range of 5 35 µg/dL 1
• Translates approximately to a 60 increase in
  developmentally disabled children and 40
  decrease in gifted children 2
• There is evidence that the reduction in IQ is
  greatest per µg/dL when lead levels are below 10
  µg/dL 6
• Children who have blood lead levels as low as 2
  µg/dL are four times as likely to be diagnosed
  with and treated for ADHD 7

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Whole Blood is the Lead Test Standard

• 95 of lead in blood is associated with the red
  blood cells 8
• It is the found in hemoglobin and the cell
  membrane
• The remainder is located in the blood plasma
• To test, the cells are lysed, and lead content is
  measured by a CDC approved method
• Graphite-furnace atomic absorption spectroscopy
  (7105) 9
• ICP-MS (7300) 10
• Capillary blood is used for screens venous
  blood is considered the definitive test
• Current CDC approved lead whole blood tests are
  only considered accurate at levels more than 10
  µg/dL
• According to the CDCs website, children cannot
  be accurately classified as having blood lead
  levels above or below a value less than 10 µg/dL
  because of the inaccuracy inherent in laboratory
  testing. 11
• In children, a whole blood lead level of 10 µg/dL
  is the CDC-recognized level of action

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Other Methods of Lead Testing

• If an alternative lead test was deemed as
  accurate as a whole blood test above 10 µg/dL and
  also accurately measures body lead below 10
  µg/dL, then researchers would be able to
  determine what effects, if any, lower doses of
  lead have
• This could lead to a lowering of the current
  level of action for children

10
Promising Saliva Lead Testing Studies

• Lead in saliva correlates more with lead levels
  in blood plasma than with levels in blood 12
• Gonzalez et al. 1997 suggest that saliva is a
  good lead monitoring medium as their measured
  levels correlated to recent exposure in Mexico
  City 14
• Timchalk et al. at Pacific Northwest Laboratory
  showed in 2006 that there is a strong correlation
  in lead in saliva vs. plasma (r20.922) in lead
  acetate dosed rats 8
• Koh and Koh, 2007, state that blood contamination
  of saliva may prove to be a potential problem,
  but that saliva could be used to monitor lead in
  higher levels of exposure (gt50 µg/dl) 15

11
Less Promising Saliva Lead Testing Studies

• Barbosa et al. in 2006 showed poor correlations
  between lead in saliva vs. blood (r0.277,
  P0.008) and lead in saliva vs. plasma (r0.280,
  P0.006) 16
• Age and gender did not affect lead saliva levels
  or the lead-saliva/lead-plasma ratio
• Collected only parotid saliva
• Wilhelm et al. in 2002 showed that saliva is not
  a good medium for measuring lead in children 17
• Collected saliva with no stimulation
• No reference materials were available
• Lead saliva tests have had trouble with
  consistency

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The WSL Saliva Lead Test

• The WSL Saliva Lead Test uses citric acid to
  induce serous salivation, allowing for a
  consistent measurement
• Filter paper strips are dosed with citric acid
  via soaking in super-saturated lemonade mix
• Sample collection consists of the donor holding
  the filter in their mouths until it is soaked
  through
• It is then placed in an air-tight container until
  analysis can be performed
• Highly trained personnel are not necessary for
  sample collection
• The filter paper strips, which hold a known
  volume of saliva, are digested in 20 HNO3
• The resulting solution is filtered and run on
  ICP-MS
• ICP-MS has a detection limit less than 0.1 ppb
  for lead, whereas GFAAS has a detection limit of
  2 ppb 6,9
• ICP-MS also has a low chance of interference

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The North Carolina Study

• People who came in for mandatory lead testing and
  nutritional screening in Guilford County, NC in
  October 2005 gave venous whole blood and saliva
  samples at the same time
• Collectors from Patriot Drug Testing gathered the
  saliva samples
• Lead analysis from venous whole blood was
  performed by the North Carolina State Lab
• Lead from saliva was measured by Western Slope
  Lab employees on PE Elan DRCe ICP-MP at a Perkin
  Elmer laboratory in Chicago, IL
• Filter paper strips were digested in 20 HNO3 in
  a Hot Block Digestion Bath

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Results from the North Carolina Study

• We know from an F-test that the sample variances
  are the same
• Hence, we may use a t-test to investigate the
  null hypothesis
• We find that the means are the same within 97.5
  confidence interval for a two-tail distribution
• All samples in the study had values measured by
  both WSL and the NC State Laboratory as less than
  10 µg/dL
• Given that the venous blood test is not
  considered accurate at such lead levels, the
  results are promising

15
Further Studies Are Required

• The data do not include samples that were
  measured as being above 10 µg/dL by WSL or the NC
  State Laboratory
• In order to demonstrate the agreement of whole
  blood and WSL Lead Saliva tests, samples above 10
  µg/dL must be measured with both methods
• The difference in lead levels between the paired
  samples is in all cases less than 4 µg/dL
• If the variance at higher lead levels is the same
  as at the low lead levels measured, then the WSL
  saliva test holds promise for good agreement with
  whole blood tests
• More results may indicate that whole blood and
  the WSL saliva method show 11 correlation at
  concentrations above 10 µg/dL

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Acknowledgements

• The Centers for Disease Control and Prevention
• The children and families who participated in the
  North Carolina Study
• The North Carolina State Laboratory
• Duane Fouts, R.Ph., Erik Cissell, and all others
  who helped in sample collection and analysis
• Perkin-Elmer

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References

• Curtis D Klaasssen, ed. Casarett and Doulls
  Toxicology The Basic Science of Poisons. 5th
  ed. New York McGraw-Hill, 1996.
• Jonathon Sher. Ontario lead laws faulted. The
  London Free Press June 5, 2007, final ed B1.
• Lead poisioning. nsc.org . 14 Aug 2008.
  National Safety Council. 14 Aug 2008
  http//www.nsc.org/resources/issues/lead.aspx.
• Alan B. Bloch, Lisa R. Rosenblum, Anne M.
  Guthrie. Recommendations for Blood Lead
  Screening of Young Children Enrolled in Medicaid
  Targeting a Group at High Risk. MMWR. 8 Dec,
  2000. CDC. 14 Aug 2008 http//www.cdc.gov/mmwR/p
  review/mmwrhtml/rr4914a1.htm.
• General Lead Information Questions and
  Answers. NCEH. 14 Aug 2008. CDC. 14 Aug 2008
  http//www.cdc.gov/nceh/lead/faq/about.htm.
• Matthew M. Matusiak. Analysis of lead in whole
  blood using the Thermo Electron X Series ICP-MS.
  The Internet Journal of Laboratory Medicine.
  2006 Volume 1, Number 2. 7 Aug 2008
  http//www.ispub.com/ostia/index.php?xmlFilePathj
  ournals/ijlm/vol1n2/lead.xml.
• Joe M. Braun, Robert S. Kahn, Tanya Froehlich,
  Peggy Auinger, and Bruce P. Lanphear. Exposures
  to Environmental Toxicants and Attention Deficit
  Hyperactivity Disorder in US Children.
  Environmental Health Perspectives. V 114 No 12
  (2008) 1904-09.
• C. Timchalk, Y. Lin, K.K. Weitz, H. Wu, R.A.
  Gies, D.A. Moore, W. Yantasee. Disposition of
  Lead (Pb) in salica and blood of Sprague-Dawley
  rats following a single or repeated oral exposure
  to Pb-acetate. Toxicology. 222 (2006) 86-94.
• Schlecht, P.C. O'Connor, P.F. Eds. LEAD by
  GFAAS. NIOSH Manual of Analytical Methods, 4th
  ed. 1994 7105. 10 Aug 2008. http//www.cdc.gov/
  niosh/nmam/pdfs/7105.pdf.
• Schlecht, P.C. O'Connor, P.F. Eds. ELEMENTS by
  ICP. NIOSH Manual of Analytical Methods, 4th
  ed. 1994 7300. 10 Aug 2008. http//www.cdc.gov/
  niosh/nmam/pdfs/7300.pdf.
• Why not change the blood lead level of concern
  at this time? NCEH. 14 Aug 2008. CDC. 14 Aug
  2008 http//www.cdc.gov/nceh/lead/faq/changeBLL.ht
  m.
• Fernando Barboa Jr., José Eduardo Tanus-Samtps.
  Raquel Fernanda Gerlack, Patrick J. Parsons. A
  Criticial Review of Biomarkers Used for Monitorin
  Human Exposure to Lead Advantages, Limitations,
  and Future Needs. Environmental Health
  Perspectives. V 13 No 12 (2005) 1669-74
• Ingvar A. Bergdahl, Lars Gerhardsson, Ingrid E.
  Liljelind, Leif Milsson, Staffan Skerfving.
  Plasma-Lead Concentration Investigations into
  its Usefulness for Biological Monitoring of
  Occupatoinal Lead Exposure. American Journal of
  Industrial Medicine. 49 (2006) 93-101
• M. Gonzalez, J.A. banderas, A. Baez, R. Belmont.
  Salivary lead and cadmium in a young population
  residing in Mexico city. Toxicology Letters.
  93 (1997) 55-64
• David Soo-Quee Koh, Gerald Choon-Huat Koh. The
  use of salivary biomarkers in occupational and
  environmental medicine. Occupational
  Environmental Medicine. 64 (2007) 202-210
• Fernando Barbosa Jr., Maria Heloísa Corrêa
  Rodrigues, Maria R. Buzalaf, Francisco J. Krug,
  Raquel F. Gerlach, José Eduardo Tanus-Santos.
  Evaluation of the use of salivary lead levels as
  a surrogate of blood lead or plasma lead levels
  in lead exposed subjects. Archives of
  Toxicology. 80 (2006) 633-637
• M. Wilhelm, A. Pesch, U. Rostek, J. Beberow, N.
  Schmitz, H. Idel, U. Ranft. Concentrations of
  lead in blood, hair and saliva of German children
  living in three different areas of traffic
  density. TheScience of the Total Environment.
  297 (2002) 109-118

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Western Slope Laboratory, LLC

• Founded in 2002 by Chairman Dr. David Schneider,
  Ph.D.
• Pharmacologist
• Assoc. Prof. Pharmacology, Wayne State
  University School of Medicine, Detroit, MI
• Became 1st wholly-owned subsidiary of Coventry
  Diagnostics, LLC after re-structuring
• Located in Troy, MI
• WSL primarily tests for drugs of abuse, alcohol
  metabolites and steroids in saliva and/or urine

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Coventry Diagnostics, LLC

• Coventry is a research company focused on
  discovering and developing novel solutions using
  saliva-based diagnostics
• Coventry has many ongoing research investigations
• Metabolism of cancer agents for molecular biology
  studies, clinical trials, and dosing purposes
• Metabolism and dosing of delirium and
  antipsychotics
• Genetics, metabolism, and dosing of warfarin
• Testing for lead and other heavy metals
• Coventry spins off production companies such as
  Western Slope Laboratory, LLC

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• Where Applied and Pure
• Research
• Becomes Reality