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Amino Acid Quantitation:
Diagnosis of Inborn Errors of Metabolism and Nutritional Deficiencies
Julie Ann Neidich, MD
Medical Director
Biochemical Genetics and Cytogenetics
Quest Diagnostics Nichols Institute
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Amino Acids and Medicine
• 1902: Garrod: First application of Mendel’s concept of a gene to a human disorder, Alkaptonuria
• Clinical diagnosis: black matter in urine that has been exposed to air
• Early 1900’s: Fischer: amino acids are bound together to make polypeptides and proteins
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History of Amino Acid Assays 1
• 1952: Martin & Synge: Nobel Prize, Chemistry: partition chromatography
• Synge then used chromatography columns packed with starch to sequence amino acids in peptides
• 1958: Stein & Moore: first automated amino acid analyzer, partnered with Beckman
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History of Amino Acid Assays 2
• Time to analyze markedly reduced:– 1946 months– 1950 weeks– 1958 a day with first Beckman automated
analyzer– Improved resins in column: 4 hours– Improved data output, increased sensitivity: ~2
hours
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History of Amino Acid Assays 3
• No real change, until now….
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Why Change Platforms?
• IMPETUS FOR CHANGE: Replace the retiring Beckman 6300s for which Beckman was no longer providing support
• Enable one platform for all sample types: plasma, urine, CSF
• Decrease turn-around or anxiety time
• Retire qualitative amino acid assay
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Current Methods
• Ion Exchange Chromatography– Beckman Amino Acid Analyzer– Biochrom Amino Acid Analyzer
• HPLC
• LC/MS
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Amino Acid Analysis in the Nichols Institute Biochemical Genetics
Laboratory
• All samples run on LC/MS
• Sample types include plasma, urine and CSF
• Tests vary from single amino acid assays like urine cystine to 47 amino acid full panel
• No qualitative assays any more
• All amino acid tests are quantitative
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Plasma Amino Acids: Current Methods vs. New Method (1)
HPLC– Run time: 80 min
Amino Acid Analyzers• Biochrom
– Run time: 165 min
• Beckman 6300– Run time: 90-150 min
LC/MS
– Run time: 25 min
(no column switcher) – 21.5 min
(with column switcher)– Single analyte: 5 min
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Chromatogram from Old HPLC Method:Part One (2 – 42 minutes)
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Chromatogram from Old HPLC Method: Part Two (42 – 72 minutes)
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Myths of LC/MS
• “LC/MS is not routine”
• “Method only produces molecular weight
information”
• “LC/MS is not sensitive”
• “LC/MS is not quantitative”
• “LC/MS is not cost-effective”
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Interfacing HPLC to MS
• HPLC
• High pressure liquid phaseseparation
• No mass range limitation
• Can use inorganic buffers
• MS
• High vacuum required
• Tolerates limited gas load
• Elevated temperatures
• Depends on m/z and analyzer
• Prefers volatile buffers
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Benefits of LC/MS
For the chromatographer• Complements existing LC detectors• Does not depend on particular functional group• Can be used as a mass-specific detector• Provides both qualitative and quantitative information
For the mass spectrometrist• Can analyze compounds not amenable to GC (large, polar, thermally labile)• Allows direct coupling of LC separation; produces better information faster than "offline" LC/MS• Automates probe analysis via flow injection
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Advantages• Softest ionization available• LC/MS interface with best sensitivity• Extends mass range for multiply charged analytes• Works with a wide range of medium to high polarity compounds• Low maintenance
Disadvantages• Solution chemistry influences ionization process• Works less well with nonpolar analytes• Adduct ions (other than M+H) possible with some analytes• Some sensitivity loss at higher flow rates (~1 ml/min)
Electrospray LC/MS
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API-Electrospray Ionization
Evaporation
Rayleigh
LimitReache
d
CoulombExplosio
nAnalyte IonEvaporatio
n
++++++
++
+++
-- -- -- ++++++
+++
++-- -- --
+++++++++
++-- -- --
+++++++++
++-- -- --
+++
++
+----
+++
++
+----
+++
++
+----
+++++
+----
+++ +++- ---
+++ +++ ----
+
++++++
++
+++
-- -- -- ++++++
+++
++-- -- -- ++++++
+++
++-- -- --
+++
++
+----
++
++
+++
+ + + + + + + + + + + + +
+
+
Heated nitrogen drying gas
Dielectric capillary entrance
Nebulizer (gas shown in red)
Solvent spray
Electrospray Ions
-5,000 V
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Capillary
API Electrospray LC/MS: Spray, Ionize, Evaporate
HPLC inlet
Nebulizer
Waste
heated N2
Nebulizer gas inlet
Fragmentation zone (CID)
Neutral Molecules
Analyte IonsClusters
Salts
Skimmers
Octopole Quadrupole
HED detector
+
++
++
Lenses
++ ++ + + ++ + + +++
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• Molecular fragmentation by ion collisions with nitrogen molecules in ion optics
• Provides structural information for qualitative analysis
• Provides confirmatory ions for quantitative analysis
• Controlled via a single ion optics parameter -- "fragmentor"
What is CID (Collision Induced Dissociation)?
M n
[M ]
1+
[M ] 2+
DETAIL
Capillary
Fragmentation Zone
(CID)
Skimmers
Octopole
Lenses
Quadrupole
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Example applications: Combinatorial chemistrySynthesis and purificationAssay and methods developmentProcess development and researchStability and formulation studies
Single Quadrupole LC/MSStrengths• Low cost, less than
MS/MS• Robust, simple operation• Tolerant of non-volatile
salts and background ions• Easy to use• High sensitivity in SIM
mode
Limitations• Lack of MS/MS specificity
(can use CID)• Lack of accurate mass
information• Slower scan speed than trap
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Plasma Amino Acids: Current Method vs. New Method
HPLC– Column only good
for 200-250 injections.
– High cost of columns.
– High cost of mobile phase.
– Many interfering substances.
LC/MS– Column good for
>300 injections.
– Lower cost of columns. – Lower cost of mobile
phase.– More precision, few
interfering substances.
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Pre-Assay
2 Basic Steps:• Drying step
• Coupling step
Makes the phenylthiocarbamyl (PITC) derivatives that are actually analyzed
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Post-Assay
• Check each peak
• Create report
• Interpret report– All interpretations for amino acid panels are
signed by ABMG certified biochemical geneticists at Quest Diagnostics.
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Changes in Samples:
Newborn Screening and Follow-Up Studies
• Increased numbers of samples from infants at risk for disorders now added to the expanded newborn screen.
• Infant samples are often small volumes.
• Turn-around time is key!
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Screening Tests
• Population-based testing
• Used to identify neonates at risk for disorders before they become ill
• Usually inexpensive compared to confirmatory tests
• Higher level of false positives and potential for false negatives
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Screening vs Confirmation
ScreeningScreening: simple way to identify individuals in a large group who may have an increased risk to have any given condition.
• In general, genetic screening focuses on specific populations at increase risk for a disease based on family history, age, or geoethnic background.
• As knowledge and technology moved forward, screening for specific genetic diseases has become part of pediatric and obstetric practice.
ConfirmatoryConfirmatory: definitive and specific testing for a condition.
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How to Choose What Disorders to Screen?
• 1994 Institute of Medicine Committee on Assessing Genetic Risks:
• “Newborn screening should only take place – 1) for conditions for which there are indications of clear
benefit to the newborn, – 2) when a system is in place for confirmatory diagnosis,
and – 3) when treatment and follow-up are available for
affected newborns…”
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OK, So How to Choose?
• Public health experts have a mathematical formula for showing public health impact: Prevalence X Severity X Effectiveness of intervention = Public Health Impact
• Need to demonstrate cost utility, showing benefit in quality-adjusted years of life plus decreased public health impact
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Really, HOW TO CHOOSE?
• Pick disorders for which early treatment is clearly beneficial and
• For which you have a window of opportunity for instituting treatment and
• Which have a significant prevalence among the group tested
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Logistics of Newborn Screening
• Heel stick blood spots drawn before infant is discharged from hospital
• Best done on day 2 of feeding, to make sure there are abnormal metabolites
• With early discharge, should be repeated
• Primary care provider and referral center contacted for abnormals to secure definitive testing for confirmation of diagnosis before treatment
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Results: Possible Outcomes
1) True Positives Require continuing special services
2) False Positives No further follow-up (See 4)
3) False Negatives*** PCPs alert for unusual presentations
4) True Negatives Routine care for normal babies
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National Newborn Screening Status Report
Updated 07/18/07
The U.S. National Screening Status Report lists the status of newborn screening in the United States. Dot "" indicates that screening for the condition is universally required by Law or Rule and fully implementedA = universally offered but not yet required, B = offered to select populations, or by request, C = testing required but not yet implementedD = likely to be detected (and reported) as a by-product of MRM screening (MS/MS) targeted by Law or Rule
STATE
Core1 Conditions
Additional Conditions Included in Screening Panel (universally required
unless otherwise indicated)Hearing Endocrine Hemoglobin Other
HEAR CH CAH Hb S/S Hb S/A Hb S/C BIO GALT CF
Alabama
A
Alaska
Arizona
A C
Arkansas
CaliforniaB HHH; PRO; EMA
Colorado
B
Connecticut B HHH; HIV 2 ; NKH
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History of Newborn Screening
• Phenylketonuria (PKU)– First newborn screening test in 1960’s– Promoted by father of affected child– 1:10,000-25,000 individuals affected– Treatment with low phenylalanine diet prevents
mental retardation– Screening done by Guthrie method– Now screened in all 50 states
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History Part 2
• Congenital hypothyroidism– 2 weeks to start replacement therapy to
prevent neurological damage– 1:4000 affected – Screened in all 50 states
• Galactosemia– 1:60,000-80,000 affected– Screened in 47 states and DC
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History Part 3
• Sickle Cell Disease– 1987 NIH recommendation to screen all
newborns for Hgb SS– 1:400 African Americans affected – 1:10 African Americans carriers– All newborns screened in 41 states and DC– Only African Americans screened in 5 states, 4
states do not offer screening at all
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What Then Changed Newborn Screening?
• Advances in genetics and technology, like the ability to use tandem mass spectroscopy to quantitate a large number of analytes from a blood spot
• Advances in genetic treatment for a variety of disorders
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AAP State-by-State Survey - July 2000
• Survey of all 50 states and DC• Web address:
www.aap.org/advocacy/archives/augscreenreport.htm
• Most states screen for 4 to 5 diseases only
• Most extensive-Massachusetts (11)
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AAP Press Release - August 7, 2000
• “Newborn Screening Report Addresses Inconsistencies and Controversies” calls for nationwide standards
• No states use the most modern methods for screening
• State-to-state differences mean some children with inborn errors will die due to birth in the “wrong” state
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Copyright ©2000 American Academy of Pediatrics Pediatrics 2000;106:389-422
U.S. Newborn Screening Circa 2000
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September 2000: First Newborn Screening Awareness Month
• Promoted by 16 support groups for parents with children who have or have died of metabolic diseases and surviving patients– Tyler for Life www.TylerforLife.com, now savebabies.org
– Children's PKU Network, Florida PKU Parents, Kansas PKU Network,, Mid Atlantic Connection for PKU & Allied Disorders, National Coalition for PKU & Allied Disorders, New England Connection for PKU & Allied Disorders, PKU Organization of Wisconsin, PKU Parents of California, and Western NY PKU Association.
– Mitochondrial Disease Outreach Center, FOD Family Support Group– Magic Foundation– MSUD Family Support Group, The Organic Acidemia Association – Parents of Galactosemic Children– National Urea Cycle Disorder Foundation
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Newborn Tyler Mize
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New Trends in Newborn Screening: Technology
• Tandem mass spectroscopy for 25-30 or more diseases offers comprehensive screening for risk of 1:1200 to 1500 of true positives– Commercial labs: Pediatrix
(formerly NeoGen) in Pennsylvania with license to Mayo– Not-for-profit labs: Baylor in Dallas
(Drs. Roe and Sweetman)
• Instituted by individual states, from NJ to CA• Done from dried blood spots• Turn around time similar to regular screening
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Potential Disorders: Amino Acid Abnormalities
• Argininemia• ASA lyase deficiency• Biopterin disorders (4)• Citrullinemia 1 and 2• Homocystinuria/CBS deficiency• Hypermethioninemia• PKU and variants• MSUD• Tyrosinemia 1-3 and transient
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Potential Disorders: Organic Acid Abnormalities
• 2-methylbutyryl-CoA dehydrogenase deficiency• 3-OH-3-methylglutaryl-CoA lyase deficiency• 3-methylcrotonyl-CoA carboxylase deficiency• 3-methylglutaconic aciduria 1-4• Beta-ketothiolase deficiency• Glutaric aciduria 1• Isobutyryl-CoA dehydrogenase deficiency• Isovaleric acidemia• Methylmalonic acidemia/cobalamin defects• Propionic acidemia• Multiple carboxylase deficiency
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Potential Disorders: Fatty Acid Oxidation Abnormalities
• Carnitine transporter deficiency• Carnitine-acylcarnitine translocase deficiency• Carnitine palmitoyl transferase deficiency 1 and 2• Long chain OH-acyl-CoA dehydrogenase deficiency
(LCHAD)• MCAD deficiency• Multiple acyl-CoA dehydrogenase deficiency
(MADD/glutaric acidemia 2)• SCAD deficiency• Trifunctional protein deficiency• VLCAD deficiency
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Potential Disorders: More
• 2-methyl-3-OHbutyryl-CoA dehydrogenase deficiency
• 5-oxoprolinuria
• Ethylmalonic encephalopathy
• Homocitrullinuria-hyperornithinemia-hyperammonemia (HHH)
• Gyrate atrophy of the choroid and retina
• Malonic aciduria
• Non-ketotic hyperglycinemia
• Prolinemia 1 and 2
• Biotinidase deficiency
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Biochemical Genetics Laboratory Test Panel
• Plasma amino acid quantitation
• Urine organic acid quantitation
• Acylcarnitine profile
• Carnitine levels
• Lactate and pyruvate
• Cellular-based enzymatic assays or DNA analyses
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California Newborn Screening Follow-Up Program
• Expanded newborn screening program to begin in California on 7/1/2005
• ~600-700,000 births per year
• ~1:1500 infants with inborn errors
• Quest Diagnostics-Nichols Institute awarded the contract to provide follow-up testing for all abnormal screens
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Nutritional Disorders
• Many other conditions may cause abnormal amino acids
• Primary problems due to malabsorption or renal disorders
• Secondary problems due to poor intake, including anorexia
• Monitoring of nutritional therapy
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Nutritional Disorders: More
• Gastrointestinal malabsorption
• Short gut syndromes
• Individuals on hyperalimentation or total parenteral nutrition– Infants, including prematures– Elderly
• Cancer patients
• Renal Fanconi syndrome
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Other Nutritional Issues
• Naturipathic therapies
• Alternative medicine
• Treatment of adult disorders via amino acid therapy, including depression and hypertension
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Summary
• New LC/MS methodology developed for amino acids analysis
• Decreased run time means decreased turn around or anxiety time
• Improved accuracy of analysis
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Medical & Scientific Staff
• Senior Scientist: Scott Goldman
• Medical Director of Genetics: Charles Strom, MD, PhD
• Medical Director: Julie Neidich, MD
• Scientific Director: Denise Salazar, PhD
• Associate Scientific Director: Renius Owen, PhD
• Associate Scientific Director: Rajesh Sharma, PhD
• Genetic Counselor: Raynah Lobo, MS
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Contact Information
• Julie Neidich, MD
• Medical Director
• Biochemical Genetics and Cytogenetics
• Quest Diagnostics-Nichols Institute
• 33608 Ortega Highway
• San Juan Capistrano, CA 92675
• 949-728-4936
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Assay Information
• Amino Acids Analysis for MSUD, LC/MS, Plasma– Includes: valine, isoleucine, leucine, alloisoleucine
• Test Code: 19779X
• Specimen Requirements:– 2 mL sodium heparin (green-top) plasma, frozen
• Clinical use:– Amino Acid analysis for MSUD is necessary for the
diagnosis of inborn errors of metabolism maple syrup urine disease. The assay is also key for the continued monitoring of treatment plans for these disorders and useful for assessing nutritional status of patients.
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Assay Information
• 767X Amino Acid Analysis, LC/MS, Plasma– Includes: Aspartic Acid, Glutamic Acid, Hydroxyproline, Serine, Asparagine, Alpha-Amino
Adipic Acid, Glycine, Glutamine, Sarcosine, Beta-Alanine, Taurine, Histidine, Citrulline, Arginine, Threonine, Alanine, 1-Methylhistidine, Gamma-Amino Butyric Acid, 3-Methylhistidine, Beta-Amino Isobutyric Acid, Proline, Ethanolamine, Alpha-Amino Butyric Acid, Tyrosine, Valine, Methionine, Cystathionine, Isoleucine, Leucine, Homocystine, Phenylalanine, Tryptophan, Ornithine, Lysine
• 1776X Amino Acid Analysis, Limited, LC/MS, Plasma– Includes: Tyrosine, Valine, Isoleucine, Leucine, Phenylalanine, Tryptophan
• 19779X Amino Acid Analysis for MSUD, LC/MS, Plasma – Includes: Valine, Isoleucine, Leucine, Alloisoleucine
• 36183X Amino Acid Analysis, LC/MS, Urine– Includes: Creatinine, Aspartic Acid, Glutamic Acid, Hydroxyproline, Serine, Asparagine, Alpha-
Amino Adipic Acid, Glycine, Glutamine, Sarcosine, Beta Alanine, Taurine, Histidine, Citrulline, Arginine, Threonine, Alanine, 1-Methylhistidine, Gamma-Amino Butyric Acid, 3-Methylhistidine, Beta-Amino Isobutyric Acid, Proline, Ethanolamine, Alpha-Amino Butyric Acid, Tyrosine, Valine, Methionine, Cystathionine, Isoleucine, Leucine, Homocystine, Phenylalanine, Tryptophan, Ornithine, Lysine, Cystine, Hydroxylysine
• 29881X Amino Acid Analysis, LC/MS, CSF – Includes: Aspartic Acid, Glutamic Acid, Hydroxyproline, Serine, Asparagine, Alpha-Amino
Adipic Acid, Glycine, Glutamine, Sarcosine, Beta-Alanine, Taurine, Histidine, Citrulline, Arginine, Threonine, Alanine, Gamma-Amino Butyric Acid, Beta-Amino Isobutyric Acid, Proline, Alpha-Amino Butyric Acid, Tyrosine, Valine, Methionine, Isoleucine, Leucine, Homocystine, Phenylalanine, Tryptophan, Ornithine, Lysine