Amino Acid Quantitation - Diagnosis of Inborn Errors of Metabolism

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 www.nicholsinstitute.com 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 www.nicholsinstitute.com 2 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 www.nicholsinstitute.com 3 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 www.nicholsinstitute.com 4 History of Amino Acid Assays 3 • No real change, until now…. www.nicholsinstitute.com 5 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 www.nicholsinstitute.com 6 Current Methods • Ion Exchange Chromatography – Beckman Amino Acid Analyzer – Biochrom Amino Acid Analyzer • HPLC • LC/MS www.nicholsinstitute.com 7 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 www.nicholsinstitute.com 8 Plasma Amino Acids: Current Methods vs. New Method (1) LC/MS – Run time: 25 min 80 min Amino Acid Analyzers (no column switcher) – 21.5 min • Biochrom (with column switcher) – Run time: 165 min – Single analyte: 5 min HPLC – Run time: • Beckman 6300 – Run time: 90-150 min www.nicholsinstitute.com 9 Chromatogram from Old HPLC Method: Part One (2 – 42 minutes) www.nicholsinstitute.com 10 Chromatogram from Old HPLC Method: Part Two (42 – 72 minutes) www.nicholsinstitute.com 11 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” www.nicholsinstitute.com 12 Interfacing HPLC to MS • HPLC • High pressure liquid phase separation • 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 www.nicholsinstitute.com 13 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 www.nicholsinstitute.com 14 Electrospray LC/MS 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) www.nicholsinstitute.com 15 API-Electrospray Ionization Nebulizer (gas shown in red) Electrospray Ions Heated nitrogen drying gas -5,000 V Solvent spray + + + + + + + + + + + + + + + + + + + + + + Dielectric capillary entrance Rayleigh Limit Reached Evaporation Coulomb Explosion -+-++ + ++ ++ + +++ Evaporation Analyte Ion + + -+ + +- - + +- -+ + - + ++ +-+ + + - -+ + - - -+ + + ++ + ++ -+- + + - ---+ +--+ + + + +++ ++ + ++- + + - ---+ +-++ + -+ + ++ + ++ + +---+ +++ ++ -+ +++ + -+ + +- - + +- -+ + - + ++ +-+ +- + + - ---+ + + + ++ ++ +- + + -----+ + ++++ -+-++ ++ + www.nicholsinstitute.com 16 API Electrospray LC/MS: Spray, Ionize, Evaporate HPLC inlet Skimmers Lenses HED detector Nebulizer gas inlet Nebulizer Capillary Octopole Quadrupole + Neutral Molecules Analyte Ions Clusters Salts + + + + + + + + + + + + + + + + + heated N2 Waste Fragmentation zone (CID) www.nicholsinstitute.com 17 What is CID (Collision Induced Dissociation)? Skimmers • Octopole Molecular fragmentation by ion collisions with nitrogen molecules in ion optics Capillary • Provides structural information for qualitative analysis Provides confirmatory ions for quantitative analysis Controlled via a single ion optics parameter -- "fragmentor" M n Fragmentation Zone (CID) Lenses Quadrupole • • DETAIL [M 1]+ [M 2 + ] www.nicholsinstitute.com 18 Single Quadrupole LC/MS Strengths • 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 Example applications: Combinatorial chemistry Synthesis and purification Assay and methods development Process development and research Stability and formulation studies www.nicholsinstitute.com 19 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. www.nicholsinstitute.com 20 Pre-Assay 2 Basic Steps: • Drying step • Coupling step Makes the phenylthiocarbamyl (PITC) derivatives that are actually analyzed www.nicholsinstitute.com 21 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. www.nicholsinstitute.com 22 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! www.nicholsinstitute.com 23 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 www.nicholsinstitute.com 24 Screening vs Confirmation Screening: 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. Confirmatory: definitive and specific testing for a condition. www.nicholsinstitute.com 25 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…” www.nicholsinstitute.com 26 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 www.nicholsinstitute.com 27 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 www.nicholsinstitute.com 28 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 www.nicholsinstitute.com 29 Results: Possible Outcomes 1) True Positives Require continuing special services No further follow-up (See 4) 2) False Positives 3) False Negatives*** PCPs alert for unusual presentations 4) True Negatives Routine care for normal babies www.nicholsinstitute.com 30 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 implemented A = universally offered but not yet required, B = offered to select populations, or by request, C = testing required but not yet implemented D = likely to be detected (and reported) as a by-product of MRM screening (MS/MS) targeted by Law or Rule Core1 Conditions STATE Hearing HEAR Endocrine CH CAH Hb S/S Hemoglobin Hb S/A Hb S/C BIO Other GALT CF Additional Conditions Included in Screening Panel (universally required unless otherwise indicated) Alabama A Alaska         Arizona         A Arkansas        C  California B Colorado              HHH; PRO; EMA www.nicholsinstitute.com B Connecticut              HHH; HIV 2 ; NKH 31    B 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 – – – – www.nicholsinstitute.com 32 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 www.nicholsinstitute.com 33 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 www.nicholsinstitute.com 34 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 www.nicholsinstitute.com 35 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) www.nicholsinstitute.com 36 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 www.nicholsinstitute.com 37 U.S. Newborn Screening Circa 2000 Pediatrics 2000;106:389-422 Copyright ©2000 American Academy of Pediatrics www.nicholsinstitute.com 38 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 www.nicholsinstitute.com 39 – – – – – Newborn Tyler Mize www.nicholsinstitute.com 40 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 www.nicholsinstitute.com 41 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 www.nicholsinstitute.com 42 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 www.nicholsinstitute.com 43 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 www.nicholsinstitute.com 44 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 www.nicholsinstitute.com 45 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 www.nicholsinstitute.com 46 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 www.nicholsinstitute.com 47 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 www.nicholsinstitute.com 48 Nutritional Disorders: More • Gastrointestinal malabsorption • Short gut syndromes • Individuals on hyperalimentation or total parenteral nutrition – Infants, including prematures – Elderly • Cancer patients • Renal Fanconi syndrome www.nicholsinstitute.com 49 Other Nutritional Issues • Naturipathic therapies • Alternative medicine • Treatment of adult disorders via amino acid therapy, including depression and hypertension www.nicholsinstitute.com 50 Summary • New LC/MS methodology developed for amino acids analysis • Decreased run time means decreased turn around or anxiety time • Improved accuracy of analysis www.nicholsinstitute.com 51 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 www.nicholsinstitute.com 52 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 julie.a.neidich@questdiagnostics.com www.nicholsinstitute.com 53 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. www.nicholsinstitute.com 54 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 Includes: Valine, Isoleucine, Leucine, Alloisoleucine Includes: Creatinine, Aspartic Acid, Glutamic Acid, Hydroxyproline, Serine, Asparagine, AlphaAmino 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 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 • 19779X Amino Acid Analysis for MSUD, LC/MS, Plasma • 36183X Amino Acid Analysis, LC/MS, Urine • 29881X Amino Acid Analysis, LC/MS, CSF – www.nicholsinstitute.com 55