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CARNITINE for FOD meeting

CARNITINE for FOD meeting. Stephen G. Kahler, M.D. Arkansas Children’s Hospital University of Arkansas for Medical Sciences July 6, 2018. DISCLAIMERS. USUAL—financial, commercial, off-label uses. How we all got here. DISCOVERY OF HUMAN CARNITINE DEFICIENCY.

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CARNITINE for FOD meeting

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  1. CARNITINE for FOD meeting Stephen G. Kahler, M.D. Arkansas Children’s Hospital University of Arkansas for Medical Sciences July 6, 2018

  2. DISCLAIMERS • USUAL—financial, commercial, off-label uses. • How we all got here.

  3. DISCOVERY OF HUMAN CARNITINE DEFICIENCY ~1976 “Systemic carnitine deficiency” Recurrent hypoglycemia; increased fat accumulation in muscle and liver. Diminished amount of ketones being produced during fasting. Excess amount of incompletely metabolized fatty acids (C6, C8, C10 Dicarboxylic fatty acids) found in urine.

  4. M.K., whose urine led to the discovery of propionylcarnitine--1982

  5. CARNITINE topics • What is it? • What does it do? • Where does it come from? • Where does it go?

  6. What is carnitine? • The locomotive at the head of the train— • Carries certain chemical compounds (molecules) around the body, and in and out of the mitochondria. • Two molecular forms (mirror images)— • L- (levo-) the natural form • D- (dextro-)—unnatural (will be present when carnitine is synthesized by a chemical process) • NO LONGER AVAILABLE, AS FAR AS I KNOW.

  7. WHAT DOES CARNITINE DO? • Brings long-chain fatty acids (16-18 carbons long) into the mitochondria, to be broken down 2 at a time. • Bring out from the mitochondria shorter molecules that are accumulating. Normally the main compound is acetylcarnitine (2 carbons attached)—a great way to move useful acetyl groups around the body. • ALWAYS IMPORTANT, BUT ESPECIALLY WHEN TRYING TO USE FATS (FASTING)

  8. BRAIN LIVER PERIPHERAL TISSUES Fasting Fuel Homeostasis KETONES GLUCOSE FATTY ACIDS AMINO ACIDS

  9. WHERE DOES CARNITINE COME FROM? • Most people make all they need. • Also diet (esp meat)—vegetarians have lower levels than meat-eaters, but not a problem. • Maybe premie babies need more than they can make?

  10. WHERE DOES CARNITINE GO? • FILTERED INTO URINE, AND REABSORBED. • SOME IS LOST IN THE URINE EVERY DAY—NO MORE THAN 5% NORMALLY. MUCH LESS IF THE PLASMA (BLOOD) CARNITINE LEVEL IS LOW.

  11. WHERE IS CARNITINE? • MAINLY IN MUSCLE, HEART, AND LIVER. • BUT WE USUALLY MEASURE ONLY BLOOD AND SOMETIMES URINE LEVELS. • SO WE HAVE TO GUESS WHAT IS AVAILABLE IN THE TISSUES TO DO THE WORK. • IF THE PLASMA LEVEL IS LOW, TISSUES PROBABLY ARE. IF THE PLASMA IS OK BUT THE PATIENT HAS A METABOLIC DISORDER, THE TISSUE LEVEL MAY STILL BE LOW.

  12. WHAT CAN GO WRONG? • Can’t make carnitine?—Not sure if it’s been discovered. • Can’t adequately reabsorb carnitine from the urine (carnitine uptake defect—CUD--, also called transporter defect) • Can’t couple carnitine to other molecules, inside or outside the mitochondria (CPT2, CPT1) • Can’t be carried into the mitochondria (translocase defect) • Excess carnitine loss because of accumulation of something—C8 in MCAD deficiency, C3 in propionic acidemia, etc.

  13. What are acylcarnitines?Acyl = Carbon structures (chains)

  14. MS/MS Analysis How many Mercedes are on the parking lot? Mass Spectrometer 2 Collision Cell Mass Spectrometer 1 Autobahn Collision gas: Fog Various Automobiles Common Fragment Precursor of “Mercedes Star” Scan Dieter Matern, Mayo Clinic 2004

  15. Glucose Glucose-6-P PLASMA MC Fatty Acid LC Fatty Acid Carnitine 3 20 UDP-Gal UDP-Glu 1 2 21 Galactose Galactose-1-P Glucose-1-P I LC-Acyl-CoA Carnitine Glycogen 5 6 4 II LC-Acylcarnitine CoASH 7 Fructose-6-P CYTOPLASM 22 8 9 10 Fructose Fructose-1-P Fructose-1,6-bis-P 11 Alanine III LC-Acylcarnitine NADH + H+ NAD+ 12 Lactate Pyruvate MITOCHONDRION 23 14 13 19 Acyl-CoA Acetyl-CoA Oxaloacetate 24 27 15 Malate Citrate 3-Oxo- Acyl-CoA Dehydro- Acyl-CoA BETAOXIDATION 18 Fumarate KREBSCYCLE Isocitrate 26 25 Succinate Acetoacetyl-CoA 3-Hydroxy- Acyl-CoA 17 Cis-Aconitate Acetone 28 Succinyl-CoA 16 HMG-CoA Acetoacetate 30 3-OH-Butyrate 29 2-Oxoglutarate NADH + H+ NAD+

  16. Glucose Glucose-6-P PLASMA MC Fatty Acid LC Fatty Acid Carnitine 3 20 UDP-Gal UDP-Glu 1 2 21 Galactose Galactose-1-P Glucose-1-P I LC-Acyl-CoA Carnitine Glycogen 5 6 4 II LC-Acylcarnitine CoASH 7 Fructose-6-P CYTOPLASM 22 8 9 10 Fructose Fructose-1-P Fructose-1,6-bis-P 11 Alanine III LC-Acylcarnitine NADH + H+ NAD+ 12 Lactate Pyruvate MITOCHONDRION 23 14 13 19 Acyl-CoA Acetyl-CoA Oxaloacetate 24 27 15 Malate Citrate 3-Oxo- Acyl-CoA Dehydro- Acyl-CoA BETAOXIDATION 18 Fumarate KREBSCYCLE Isocitrate 26 25 Succinate Acetoacetyl-CoA 3-Hydroxy- Acyl-CoA 17 Cis-Aconitate Acetone 28 Succinyl-CoA 16 HMG-CoA Acetoacetate 30 3-OH-Butyrate 29 2-Oxoglutarate NADH + H+ NAD+

  17. Glucose Glucose-6-P PLASMA MC Fatty Acid LC Fatty Acid Carnitine 3 20 UDP-Gal UDP-Glu 1 2 21 Galactose Galactose-1-P Glucose-1-P I LC-Acyl-CoA Carnitine Glycogen 5 6 4 II LC-Acylcarnitine CoASH 7 Fructose-6-P CYTOPLASM 22 8 9 10 Fructose Fructose-1-P Fructose-1,6-bis-P 11 Alanine III LC-Acylcarnitine NADH + H+ NAD+ 12 Lactate Pyruvate MITOCHONDRION 23 14 13 19 Acyl-CoA Acetyl-CoA Oxaloacetate 24 27 15 Malate Citrate 3-Oxo- Acyl-CoA Dehydro- Acyl-CoA BETAOXIDATION 18 Fumarate KREBSCYCLE Isocitrate 26 25 Succinate Acetoacetyl-CoA 3-Hydroxy- Acyl-CoA 17 Cis-Aconitate Acetone 28 Succinyl-CoA 16 HMG-CoA Acetoacetate 30 3-OH-Butyrate 29 2-Oxoglutarate NADH + H+ NAD+

  18. Glucose Glucose-6-P PLASMA MC Fatty Acid LC Fatty Acid Carnitine 3 20 UDP-Gal UDP-Glu 1 2 21 Galactose Galactose-1-P Glucose-1-P I LC-Acyl-CoA Carnitine Glycogen 5 6 4 II LC-Acylcarnitine CoASH 7 Fructose-6-P CYTOPLASM 22 8 9 10 Fructose Fructose-1-P Fructose-1,6-bis-P 11 Alanine III LC-Acylcarnitine NADH + H+ NAD+ 12 Lactate Pyruvate MITOCHONDRION 23 14 13 19 Acyl-CoA Acetyl-CoA Oxaloacetate 24 27 15 Malate Citrate 3-Oxo- Acyl-CoA Dehydro- Acyl-CoA BETAOXIDATION 18 Fumarate KREBSCYCLE Isocitrate 26 25 Succinate Acetoacetyl-CoA 3-Hydroxy- Acyl-CoA 17 Cis-Aconitate Acetone 28 Succinyl-CoA 16 HMG-CoA Acetoacetate 30 3-OH-Butyrate 29 2-Oxoglutarate NADH + H+ NAD+

  19. Organic aciduria Galactose Glycogen Glucose-1-P Fat Glucose Glucose-6-P Glucose Acyl-CoA _ Acyl-carnitine Fructose Triose-3-P Acyl-carnitine Pyruvate Lactate Acyl-CoA NAD NADH+H+ H2O Acetyl-CoA -oxidation V.O.M.I.T. Resp. Chain Oxaloacetate Citrate Propionyl-CoA O2 PA Krebs cycle NADH+H+ MMA-CoA MMA Succinyl-CoA

  20. Acylcarnitines • Fatty Acid Oxidation Defects • MCAD, SCAD, VLCAD, LCHAD • Carnitine Palmitoyltransferase II (CPT II) • Glutaric Acidemia II • 2,4 Dienoyl-CoA Reductase Deficiency

  21. Fatty acid oxidation disorder Galactose Glycogen Glucose-1-P Fat Glucose Glucose-6-P Glucose Acyl-CoA _ Acyl-carnitine Fructose Triose-3-P Acyl-carnitine Lactate Pyruvate Acyl-CoA NAD NADH+H+ H2O Acetyl-CoA -oxidation V.O.M.I.T. Resp. Chain Oxaloacetate Citrate Propionyl-CoA O2 Krebs cycle NADH+H+ MMA-CoA Succinyl-CoA

  22. MCAD deficiency as Reye syndrome--recovered

  23. MCAD • Medium Chain Co-A Acyl-Dehydrogenase Deficiency • Incidence ~1:15,000 Northern European • 25-50% first episode fatality rate • Avoid Prolonged Fasting, + Carnitine • Requires new methodology • Tandem Mass Spectrometry for acylcarnitines

  24. MCAD Def and Northern Europe

  25. M.K., whose urine led to the discovery of propionylcarnitine--1982

  26. Propionic acidemia--crisis and recovery

  27. Glutaric Aciduria Type I--Megalencephaly +

  28. WHO NEEDS CARNITINE? • Controversies! • CUD—transporter defect. Lots, and always. • Some patients don’t seem to suffer, but may be at risk for sudden death. • MCAD deficiency, esp while young • Propionic acidemia—YES. MMA—usually not(?) • Isovalericacidemia—sometimes. • VLCAD, LCHAD deficiency? • Glutaricacidemia type I?

  29. WHEN/HOW SHOULD CARNITINE BE GIVEN? • MORE CONTROVERSIES! • MORE THAN ONCE A DAY • NOT JUST WHEN SICK • LOTS OF PROBLEMS ARE SOLVED WHEN IT IS GIVEN IV • WHAT ABOUT GIVING ACETYLCARNITINE (ACETYL-L-CARNITINE)?

  30. WHAT HAPPENS WITH CARNITINE SUPPLEMENTATION? • Body-builders • Otherwise healthy people • Mitochondrial dysfunction • Autism? • Intestinal motility problems? • Too much carnitine—exceeds intestinal ability to absorbfish odor. Also diarrhea.

  31. WHERE DOES VARIATION COME FROM?

  32. LET’S TALK ABOUT MUTATIONS--1 • Changes in DNA, the master set of instructions in all cells. • 2 copies of nearly all genes; 1 copy from each parent. • Exception—genes on the X chromosome (girls have 2, boys have 1). • Recessive means hidden—effects of a recessive mutation are hidden (sort of) if there is a normal copy as well.

  33. LET’S TALK ABOUT MUTATIONS--2 • Genes in DNA are in sections, interrupted by intervening sequences • A copy must be made from the DNA, then the intervening sequences removed (splicing the working parts of the gene together) • The mature messenger RNA must be read (translated) 3 DNA letters at a time to produce an amino acid chain (protein).

  34. LET’S TALK ABOUT MUTATIONS--3 • In our disorders, the proteins made are ENZYMES, which make chemical reactions happen faster.

  35. LET’S TALK ABOUT MUTATIONS--4 • WHAT COULD POSSIBLY GO WRONG? • Errors in DNA • Nonsense—DNA (RNA actually) can’t be read/translated • Missplaced STOP triplet (codon) • Mistakes near splice site • Missense—it works, but not all that well (or sometimes too well!) • Rate problems—not making enough of something that works, or recycling it too soon. • How many mutations can be found in 1 gene? • How many combinations of mutations leading to recessive illness?

  36. WHAT ELSE COULD GO WRONG?

  37. LET’S THINK ABOUT OTHER GENES • What about the other 20,000+ genes?

  38. LET’S THINK ABOUT ENVIRONMENTAL (= NOT GENETIC) FACTORS • When do people get sick? • Viruses • Fasting • Fever • Diet mishaps • HOW DO PEOPLE STAY LESS SICK? • GOOD NUTRITION!!! • WHAT ABOUT SUPPLEMENTS/VITAMINS?

  39. LET’S THINK ABOUT “DUAL DIAGNOSIS” • 5 – 10% of patients with a nameable disorder will have a SECOND nameable disorder that helps explain what is going on. • Sometimes the “2nd disorder” is discovered first. This can lead to confusion, as the discovered disorder is wrongfully blamed for the illness (CONTENTIOUS!)

  40. And LET’S THINK ABOUT HOW OUR TREATMENTS WORK • Same general principles— • Each treatment may have more than one effect • Each treatment may have a different effect, depending on the patient • The reason we use something may not be the same thing as the reason why it is actually working.

  41. SOME WORDS TO LIVE BY • Keep thinking about “medicine-based evidence” as the beginning of “evidence-based medicine” • “YOU CAN OBSERVE A LOT JUST BY WATCHING.” Yogi Berra • “CHERISH YOUR EXCEPTIONS.” Barton Childs, MD • “LISTEN TO YOUR PATIENT. HE’S TRYING TO TELL YOU SOMETHING.” Eugene Stead, MD

  42. Acylcarnitine analysis

  43. Rationale for analysis • 1980s: Roe & Millington investigate Reye syndrome • Multiple methods involving GC/MS needed to measure free, total, and acylcarnitines • MS/MS method allowed for a single analytic method for all analytes of interest • Done on blood/plasma now; was originally developed for urine

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