Niezen-Koning, Klaziena Elisabeth

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Proceedings - University of Groningen

   University of GroningenMitochondrial betha-oxidationNiezen-Koning, Klaziena ElisabethIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1995Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Niezen-Koning, K. E. (1995). Mitochondrial betha-oxidation: genetic deficiency and therapy. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 03-06-2022lhr istensen, E., Bross, P.,n-chain acyl CoA dehydroge-rnexpected death in childts in lysine oxidation: 2 keto-.  S ly ,  w .S. ,  Va l le .  D .  (eds)Graw-Hil l ,  New York: 1-15 1(1989) Prerratal diagnosis ofcultured amniocytes andt ,  L . ,  Wanders ,  R.J . ,  Ugar te .yacyl-CoA dehydro genaseiort-chain acyl-CoA dehydrnt with short-chain acyl-1 to the patient with a fattyof iong-chain ac1' l-CoAt r  V o l .  1 1 9  ( 1 ,  P t  l )J as a Ioading test foris  1  I  (supp l  lZ )  pp  22  t - ) .2+ .r in the newborn produce-chain acyl CoA dehydro-ing  tesr  fo r  rn td iunr - t l ia inLy ingested 3-phenyl-nd therapeutic implicationsdrogenase deficiency. Pediatrrs a probe of fatty acid:tion of endemic riboflavrn, ^ ^ ;  i -  ^ ^ ^ - l : - r - i ^ , . .i c s t  r 1 1  P d c u r d r r r L > .rtes. Eur I  Pediatr Vol. I  5 0tal and early postnatalleath. I  hrherit  Metab DisSUMMARYThe mitochondrion is the powerhouse of the cell, where energy is generated. Energycan be generated from the oxidation of proteins, polysaccharides and fats supplied byconsuming food. Sugars, fatty acids and several amino acids are converted into the acetylunit of acetyl-CoA. Acetyl-CoA is donating its acetyl units to the citric acid cycle, whereoxidatlon to CO2 is taking place. Adenosine triphosphate (ATP) is generated as electronsflow to oxygen upon oxidative phosphorylation.Thus, fatty acids are primarily available as supply from food. Secondary, fatty acids areavailable as a supply created from intracellular stores. After a period of fasting theglycogen reservoir is consumed first. When fasting lasts longer than one day, fats/íattyacids will be released frorn adipose tissue into the blood stream. Fatty acids with a chainlength of tZ to 20 carbon atoms with no ('saturated') or up to two double bonds('unsaturated') are mostly used as fuel.Uptake of fatty acids takes place very efficiently in the liver. Fatty acid binding proteins(FABPs) are presumed to play a role in uptake of fatty acids. Their proposed functions canbe summarized as follows. In the B-oxidation FABPs act as carriers for fatty acids betweenthe plasma membrane and the outer mitochondrial membrane and peroxisomalrnembrane, while they are also involved irl the regulation of the cellular concentration offatty acids and their CoA esters.Before fatty acids can be used by the cell, they will be combined first with coenzyme Ato form a highly polar thioester. Thls reaction is catalyzed by fatty acyi-CoA synthetases.When the fatty acids, specifically long-chain fatty acids, are activated to long-chain fattyacyl-CoA they can cross the mitochondrial outer rnembrane. Short and medium chain fattyacids can readily penetrate mitochondria without carrier mediated transport. They areactivated to their CoA esters in the mitochondrial matrix and subsequently degraded in thefatty acid B-oxidation. Before long-chain fatty acyl-CoA can undergo fatty acid B-oxidationthey have to be transported across the inner mitochondrial membrane. Carnitine has anessential physiological function in this process.Carnitine plays a physlological role in a number of other metabolic processes,including buffering of the mitochondrial acyl-CoA/CoA couple; scavenger system for acylgroups; peroxisomal fatty acid oxidation; intracellular communication by means of storageand transport of rnetabolic energy; facil itating the oxidation of keto acids, branched-chainamino acids and medium-chain fatty acids (actívated in cytosol or cell and not in thematrix of the mitochondrlon) and membrane stabilization.As mentioned before carnitine plays a physiological role in facil itating the oxidation oflong chain fatty acids. After activation of a long-chain fatty acid, such as palmitate, thepalmitoyl-CoA ester is transferred through the mitochondrial outer membrane into theintermembrane space without the use of the carnitine system. Transesterification topalmitoylcarnitine via a reaction catalyzed by carnitine palmitoyltransferase I (CPT 1)takes place, because the inner membrane of the mitochondrion is virtually impermeable toCoA and its derivatives. CPT I is localized on the inner aspect of the outer mitochondriaimembrane.The palmitoylcarnitine is transported across the mitochondrial inner membrane via acarnitine acylcarnitine translocase. Carnitine acylcarnitine translocase is an antiport systemwhereby palmitoylcarnitine is transported into the matrix in exchange for carnitine.1 1 1The matrix palmitoylcarnitine is a substrate for carnitine palmitoyltransferase 2 (CPT2) using matrix CoASH to form palmitoyl-CoA in the matrix, thus releasing carnitine. Thisprocess makes palmitoyl-CoA available for entering the mitochondrial fatty acidB-oxidation spiral.In mitochondrial fatty acid B-oxidation stepwise degradation of fatty acids of variouschain lengths takes place, generating the needed energy. After four sequential steps ofdehydrogenation (plus simultaneous transfer of electrons to electron transfer flavoprotein[ETF]), hydration, again dehydrogenation and thiolytic cleavage, the resulting acyl-CoAester now shortened by 2 carbon atoms, can reenter the B-oxidation spiral.Fatty acid B-oxidation is regulated by a mechanism in which interaction of CpT I andmalonyl-CoA plays a central role. Hormonal control of fatty acid B-oxidation is exerted atthe level of substrate mobilization from adipose tissue and by indirect effêcts on CPT 1. Ofrelevance are the inhibiting effect of insulin and the indirect stimulation of glucagon onB-oxidation.For the pathophysiology of fatty acid B-oxidation disorders it is essential to appreciatethe central role that fatty acids play in times of fasting. Beyond a day of fasting, glycogenreserves are depleted and alternative means are activated: gluconeogenesis, ketogenesis andfatty acid oxidation. Oxidation of fatty acids results in the end-product, acetyl-CoA, whichcan be the precursor of the ketone bodies, i.e. acetoacetate and B-hydroxybutyrate, whichrepresent an essential source of energy in brain under àsting conditions.Under fasting conditions patients with a fatty acid B-oxidation disorder develophypoketotic hypoglycemia due to an inability to generate acetyl-CoA resulting in lowlevels of ketone bodies and an inability to generate sufficient glucose. Apart from thatpresenting symptoms including lethargy leading to coma, Reye-like syndrome, acute lifethreatening event (ALTE), muscle weakness, cardiac problems, hepatomegaly andabnormal laboratory findings (hyperuricemia, hyperammonemia, dicarboxylic aciduria,acidosis, increased activity of alanine and aspartate aminotransferases and of creatinekinase and alterations in plasma or tissue carnitine concentration) can appear. However,fatty acid oxidation defects only produce abnormalities intermittendy. So during an acuteattack only the urinary organic acid proÍile and blood carnitine levels can give anindication for inherited fatty acid oxidation disorders. The study described in this thesiswas undertaken to try to shed more light upon this group of disorders.For this purpose we have developed sensitive and reliable enzyme assays for diagnosisof several fatty acid B-oxidation deficiencies including short-chain, medium-chain, and(very) long-chain acyi-CoA dehydrogenase (see chapters 2-a). The concentration of the3-hydroxy fatty acid acyl-CoA, formed from the substrate fatty acid acyl-CoA during theenzyme assay, could be measured after hydrolysis as its 3-hydroxy fatty acid. Forquantification we have used stable isotopes and gas chromatography/mass spectrometry(GC-MS).As we have mentioned before, fatty acid oxidation defects only produce intermittentabnormalities. Especially when a patient, suspected to suffêr from a defect in theB-oxidation, is in an asymptomatic phase of the disease the diagnosis of this patient can bevery difficult as is the case in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.MCAD deficiency is the most common defect of B-oxidation. Thus we have evaluated our2l patients with MCAD deficiency with respect to their clinical symptoms at presentation,laboratory findings, diagnostic and therapeutic approach (see chapter 5). Promptdiagnosis and initiation of treatment will improve long-term prognosis of a MCADdeficient (MCADD) patient. We have found that due to our family screening program inwhich first degree family members of MCADD patients are screened for MCAD, 30% ofthese asymptomatic family members (brothers and sisters) were MCADD. Thus makingI t zthe percerespectivtfrom thesuspectecacids inacid, dicain leucocIn orrB-oxidatipatient islevel ofcardioresperipheraand the ro i t l r o r  f tL r ( r r l r  5gluconeoleucocyteFurther imetabolisis a patiewho presshowed astrictly aof ketolytransferasconuast tis disturbbrain uncCultuWe devefibroblastiodide (Iinvestigatcan influrin the strskin fibrcinfluence(chapter Iwas thatpatients clactic acirusing ratperturbatfind increstatus.  W,amountsunfortun:As mrAnrr-+G tpresent isferase 2 (CPTcarnitine. Thisial fatty acidcids of variousential steps ofer flavoproteinIting acyl-CoAr of CPT I andrn is exerted ats on CPT 1. Ofrf glucagon onil to appreciate;ting, glycogen:etogenesis and:yl-CoA, rvhichrutyrate, u'hichorder developsulting in lowpart from that>me, acute lifetomegaly andxylic aciduria,rd of creatineear. However,uring an acutecan give an. in this thesis; for diagnosisLm-chain, and.tration of therA during the.tty acid. ForspectIometrye intermittentlefect in thepatient can be)) deficiency.evaluated ourpresentatron,5). Promptof a MCADg program inlAD, 30% ofThus makingthe percentage of ALTEs in our population lower compared to literature, 5% versus 2170,respectively. For genetic counselling carrier detection takes place, in which leucocytesfrom the parents of a MCADD patient are investigated. Definitive evaluation of a patientsuspected to suffêr from a MCAD deficiency should include GC-MS of urinary organicacids in order to look for specific metabolites (glycin derivatives, S-hydroxy hexanoicacid, dicarboxylic acids), determination of the carnitine status, and specific enzyme assaysin leucocvtes or cultured skin fibroblasts.In order to give an impression of the diverging presentation of different disorders ofB-oxidation we describe two patients, each with a specific defect (see chapter 6). The firstpatient is the fourth case described in literature with an unique B-oxidation defect at thelevel of the carnitine acylcarnitine carrier. The presenting clinical symptoms werecardiorespiratory insuÍÍiciency, subcomatose state and bradycardia with decreasedperipheral circulation. The combination of extreme hypoglycemia, hyperammonaemiaand the observed pattern of urinary organic acids rose suspicion to consider defects ineither gluconeogenesis, urea cycle or fatty acid B-oxidation. Defects in eithergluconeogenesis or urea cycle were ruled out. Measurement of the overall B-oxidation inleucocytes revealed a complete absence of long-chain fatty acid (paimitate) B-oxidation.Further investigations in cultured skin fibroblasts on the level of long-chain fatty acidmetabolism showed a deficiency of carnitine acylcarnitine transiocase. The second patientis a patient with a succinyl-CoA acetoacetate transferase deficiency, a defect of ketolysis,who presented early in life with recurrent ketoacidotic attacks and persistent ketonuria. Heshowed a normal development later in life up to 9 years of age. Although this defect is notstrictly a defect in mitochondrial B-oxidation, there are some similarities between a defectof ketolysis and mitochondrial fatty acid B-oxidation. In succinyl-CoA acetoacetatetransferase deÍiciency ketone body production is adequate but utilization is disturbed incontrast to a mitochondrial B-oxidation deÍèct, in which the production of ketone bodiesis disturbed. In both mitochondrial B-oxidation and ketolysis defects consequences for thebrain under fasting conditions are identical.Cultured skin fibroblasts are a highly useful source for elucidating inherited defects.We developed a screening procedure to detect B-oxidation defects in cultured skinÍibroblasts using the fluorescent dye 2-[a-(dimethylamino)styryl]-1-methylpyridiniumiodide (DASPMI) (chapter 7). This fluorescence microscopical approach is used toinvestigate whether it might provide insight as to how B-oxidation related enzyme defectscan influence the mitochondrial redox status, which presumably arises from abberationsin the structure of mitochondria. However, no abberations could be detected in culturedskin fibroblasts from MCADD patients. Apart from this approach, we have also studied theinfluence of p-oxidation related enzyme defects on the mitochondrial redox status(chapter 8). We have used cultured skin fibroblasts from MCADD patients. Our hypothesiswas that a presumed perturbation of mitochondrial structure in tissue from MCADDpatients on increased concentration of octanoate could be the basis of the sometimes mildlactic acidosis in MCADD patients. From literature we have learned that in experimentsusing rabbits that were treated with pathological concentrations of octanoate showedperturbation of mitochondrial structure in the brains were found. We were expecting tofind increased ratios of lactate/pyruvate, which provide insight in the mitochondrial redoxstatus. We have challenged cultured skin fibroblasts from MCADD patients with increasingarnounts of octanoic acid and measured the lactate/pyruvate ratios. However,unfortunately our hypothesis has not been affirmed by our standarized experiments.As mentioned before MCAD deficiency is the most common defect of B-oxidation. TheAnrr-)G transition is the most common mutation in MCAD deficiency. This mutation ispresent in 90oÁ of the patients with a MCAD deficiency. In order to determine the1 1 3frequency of Arrr->G carriers in The Netherlands a study is performed in which 6195Guthrie cards are analyzed for the presence of the Anrr-+G mutation (see chapter 9). Thesecards were obtained from the 5 PKU screening laboratories in The Netherlands. Theestimated prevalence of newborns with MCAD deficiency is I : 12,500 in The Netherlands.Inforniation concerning the prevalence together with its high morbidity and mortalitymay provide the background to consider an extended screening for this common MCADmutation in newborns Írom The Netherlands.We hope that the results of the investigations presented in this thesis and thedescription of clinical and biochemical features together with the approach to the patientsuspected for a mitochondrial B-oxidation defect, will add to the long term prognosis ofpatients suffering from this important and intriguing group of disorders.l r +

Mitochondrial betha-oxidation:genetic deficiency and therapy

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Mitochondrial betha-oxidation:genetic deficiency and therapy

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