Subclinical effects of long-chain fatty acid ß-oxidation deficiency on the adult heart: A case-control magnetic resonance study.

Cardiomyopathy can be a severe complication in patients with long-chain fatty acid ß-oxidation disorders (LCFAOD), particularly during episodes of metabolic derangement. It is unknown whether latent cardiac abnormalities exist in adult patients. To investigate cardiac involvement in LCFAOD, we used proton magnetic resonance imaging (MRI) and spectroscopy (1 H-MRS) to quantify heart function, myocardial tissue characteristics, and myocardial lipid content in 14 adult patients (two with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD); four with carnitine palmitoyltransferase II deficiency (CPT2D); and eight with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD)) and 14 gender-, age-, and BMI-matched control subjects. Examinations included cine MRI, MR tagging, native myocardial T1 and T2 mapping, and localized 1 H-MRS at 3 Tesla. Left ventricular (LV) myocardial mass (P = .011) and the LV myocardial mass-to-volume ratio (P = .008) were higher in patients, while ejection fraction (EF) was normal (P = .397). LV torsion was higher in patients (P = .026), whereas circumferential shortening was similar compared with controls (P = .875). LV hypertrophy was accompanied by high myocardial T1 values (indicative of diffuse fibrosis) in two patients, and additionally a low EF in one case. Myocardial lipid content was similar in patients and controls. We identified subclinical morphological and functional differences between the hearts of LCFAOD patients and matched control subjects using state-of-the-art MR methods. Our results suggest a chronic cardiac disease phenotype and hypertrophic LV remodeling of the heart in LCFAOD, potentially triggered by a mild, but chronic, energy deficiency, rather than by lipotoxic effects of accumulating lipid metabolites.

Uncoupling, metabolic inhibition and induction of mitochondrial permeability transition in rat liver mitochondria caused by the major long-chain hydroxyl monocarboxylic fatty acids accumulating in LCHAD deficiency.

Patients with long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiency commonly present liver dysfunction whose pathogenesis is unknown. We studied the effects of long-chain 3-hydroxylated fatty acids (LCHFA) that accumulate in LCHAD deficiency on liver bioenergetics using mitochondrial preparations from young rats. We provide strong evidence that 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, the monocarboxylic acids that are found at the highest tissue concentrations in this disorder, act as metabolic inhibitors and uncouplers of oxidative phosphorylation. These conclusions are based on the findings that these fatty acids decreased ADP-stimulated (state 3) and uncoupled respiration, mitochondrial membrane potential and NAD(P)H content, and, in contrast, increased resting (state 4) respiration. We also verified that 3HTA and 3HPA markedly reduced Ca2+ retention capacity and induced swelling in Ca2+-loaded mitochondria. These effects were mediated by mitochondrial permeability transition (MPT) induction since they were totally prevented by the classical MPT inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca2+ uptake blocker. Taken together, our data demonstrate that the major monocarboxylic LCHFA accumulating in LCHAD deficiency disrupt energy mitochondrial homeostasis in the liver. It is proposed that this pathomechanism may explain at least in part the hepatic alterations characteristic of the affected patients.

Mitochondrial bioenergetics deregulation caused by long-chain 3-hydroxy fatty acids accumulating in LCHAD and MTP deficiencies in rat brain: a possible role of mPTP opening as a pathomechanism in these disorders?

Long-chain 3-hydroxylated fatty acids (LCHFA) accumulate in long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. Affected patients usually present severe neonatal symptoms involving cardiac and hepatic functions, although long-term neurological abnormalities are also commonly observed. Since the underlying mechanisms of brain damage are practically unknown and have not been properly investigated, we studied the effects of LCHFA on important parameters of mitochondrial homeostasis in isolated mitochondria from cerebral cortex of developing rats. 3-Hydroxytetradecanoic acid (3 HTA) reduced mitochondrial membrane potential, NAD(P)H levels, Ca(2+) retention capacity and ATP content, besides inducing swelling, cytochrome c release and H2O2 production in Ca(2+)-loaded mitochondrial preparations. We also found that cyclosporine A plus ADP, as well as ruthenium red, a Ca(2+) uptake blocker, prevented these effects, suggesting the involvement of the mitochondrial permeability transition pore (mPTP) and an important role for Ca(2+), respectively. 3-Hydroxydodecanoic and 3-hydroxypalmitic acids, that also accumulate in LCHAD and MTP deficiencies, similarly induced mitochondrial swelling and decreased ATP content, but to a variable degree pending on the size of their carbon chain. It is proposed that mPTP opening induced by LCHFA disrupts brain bioenergetics and may contribute at least partly to explain the neurologic dysfunction observed in patients affected by LCHAD and MTP deficiencies.

Increased and early lipolysis in children with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency during fast.

Children with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD) have a defect in the degradation of long-chain fatty acids and are at risk of hypoketotic hypoglycemia and insufficient energy production as well as accumulation of toxic fatty acid intermediates. Knowledge on substrate metabolism in children with LCHAD deficiency during fasting is limited. Treatment guidelines differ between centers, both as far as length of fasting periods and need for night feeds are concerned. To increase the understanding of fasting intolerance and improve treatment recommendations, children with LCHAD deficiency were investigated with stable isotope technique, microdialysis, and indirect calometry, in order to assess lipolysis and glucose production during 6 h of fasting. We found an early and increased lipolysis and accumulation of long chain acylcarnitines after 4 h of fasting, albeit no patients developed hypoglycemia. The rate of glycerol production, reflecting lipolysis, averaged 7.7 ± 1.6 µmol/kg/min, which is higher compared to that of peers. The rate of glucose production was normal for age; 19.6 ± 3.4 µmol/kg/min (3.5 ± 0.6 mg/kg/min). Resting energy expenditure was also normal, even though the respiratory quotient was increased indicating mainly glucose oxidation. The results show that lipolysis and accumulation of long chain acylcarnitines occurs before hypoglycemia in fasting children with LCHAD, which may indicate more limited fasting tolerance than previously suggested.

[Expression of long chain fatty acid oxidase in maternal and fetal tissues in preeclampsia-like mouse model in mid-gestation].

OBJECTIVE: To explore the protein expression and distribution of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) in various maternal and fetal tissues in preeclampsia-like and normal pregnant mouse models during the middle trimester. METHODS: C57BL/6J mice were divided into preeclampsia-like experimental group with Nw-nitro-L-arginine-methyl ester injection and normal pregnancy control group with normal saline injection. Samples were taken at Day 14 of gestation. Immunohistochemistry was used to detect the LCHAD protein expression and distribution in various maternal and fetal tissues. RESULTS: The mean arterial pressure was (115.6 ± 3.3) mmHg (1 mmHg = 0.133 kPa) in experimental group and (96.2 ± 4.9) mmHg in control group (P < 0.05). The urine protein contents were (97.0 ± 4.3) µg/g body weight/24h in experimental group and (60.8 ± 4.9) µg/g body weight/24h in control group (P < 0.05). In both groups, LCHAD protein was expressed in maternal liver, kidney, heart and placenta tissues during the middle trimester. And maternal hepatic tissue, myocardial tissue, renal tubule and spongiotrophoblast showed a strong positive expression, placental labyrinth layer a moderate positive expression and glomerulus a weakly positive expression. LCHAD expression of maternal liver and placenta decreased significantly in experimental group (P < 0.05). At Day 14 of pregnancy, LCHAD was expressed in fetal liver, kidney, heart, lung tissue and neural retina. And renal tubule, pulmonary epithelial tissue and neural retina showed a strong positive expression, fetal liver a moderate positive expression and myocardial tissue and glomerulus a weakly positive expression. There was no significant inter-group difference in fetal tissues. CONCLUSION: LCHAD is expressed and distributed widely in maternal and fetal tissues during the middle trimester. And it may play an important role in maternal metabolism and placenta-fetus development during pregnancy. There is an abnormal protein expression LCHAD in maternal liver and placenta with preeclampsia-like changes.

[Variation of long-chain 3-hydroxyacyl-CoA dehydrogenase DNA methylation in placenta of different preeclampsia-like mouse models].

OBJECTIVE: By detecting the variation of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) DNA methylation in preeclampsia-like mouse models generated by different ways, to explore the roles of multifactor and multiple pathways in preeclampsia pathogenesis on molecular basis. METHODS: Established preeclampsia-like mouse models in different ways and divided into groups as follows: (1) Nw-nitro-L-arginine-methyl ester (L-NAME) group: wild-type pregnant mouse received subcutaneous injection of L-NAME; (2) lipopolysaccharide (LPS) group: wild-type pregnant mouse received intraperitoneal injection of LPS; (3) apolipoprotein C-III (ApoC3) group: ApoC3 transgenic pregnant mouse with dysregulated lipid metabolism received subcutaneous injection of L-NAME; (4) ß2 glycoprotein I (ß-2GPI) group: wild-type pregnant mouse received subcutaneous injection of ß-2GPI. According to the first injection time (on day 3, 11, 16 respectively), the L-NAME, LPS and ApoC3 groups were further subdivided into: pre-implantation (PI) experimental stage, early gestation (EG) experimental stage, and late gestation (LG) experimental stage. ß-2GPI group was only injected before implantation. LCHAD gene methylation levels in placental were detected in different experimental stage. Normal saline control groups were set within wild-type and ApoC3 transgenic pregnant mice simultaneously. RESULTS: (1) CG sites in LCHAD DNA: 45 CG sites were detected in the range of 728 bp before LCHAD gene transcription start site, the 5, 12, 13, 14, 15, 16, 19, 24, 25, 27, 28, 29, 30, 31, 32, 34, 35, 43 CG sites were complex sites which contained two or more CG sequences, others were single site which contained one CG sequence. The 3, 5, 6, 11, 13, 14, 18, 28 sites in L-NAME, LPS, ApoC3 and ß-2GPI groups showed different high levels of methylation; the 16, 25, 31, 42, 44 sites showed different low levels of methylation; other 32 sites were unmethylated. (2) Comparison of LCHAD gene methylation between different groups: the methylation levels of LCAHD gene at 3, 11, 13, 14, 18 sites in L-NAME, LPS, ApoC3 and ß-2GPI groups were significantly higher than those in the normal saline control group (P < 0.05); and the methylation levels of 42, 44 sites in these groups were significantly lower than those in the normal saline control group (P < 0.05). (3) Methylation of LCHAD gene at the same site between different experimental stages: ① The 3, 11, 18 sites of EG experimental stage was significantly lower than PI and LG experimental stage in L-NAME group (P < 0.05); the 3, 11, 18 sites of PI experimental stage was significantly lower than EG and LG experimental stage in LPS group (P < 0.05); these sites of PI experimental stage was significantly higher than EG and LG experimental stages in ApoC3 group (P < 0.05). ② The methylation of site 5 in L-NAME and LPS groups were significantly higher than that of the normal saline control group (P < 0.05), and the LG experimental stages were significantly higher than other stages, but in ApoC3 group, only PI and EG stages were significantly higher than the normal saline control group (P < 0.05). ③ At site 6 in L-NAME group which showed high methylation level was significantly higher than the same site in other groups which showed low methylation level (P < 0.05). ④ At 13, 14 sites, earlier preeclampsia onset caused a lower methylation level in L-NAME group, but PI experimental stage was significantly higher than EG and LG experimental stages in LPS group (P < 0.05), EG experimental stage was significantly higher than PI and LG experimental stages in ApoC3 group (P < 0.05). ⑤ At site 28, earlier preeclampsia onset caused a higher methylation level in L-NAME group, but PI experimental stage was significantly lower than EG and LG experimental stages in LPS group (P < 0.05), EG experimental stage was significantly higher than PI and LG experimental stages in ApoC3 group (P < 0.05). ⑥ The 16, 25, 31 sites in ApoC3 group were significantly higher than other groups (P < 0.05). ⑦ At site 42 in ß-2GPI group was unmethylated, but it in other groups showed low methylation level, the methylation level of site 42 in ß-2GPI group was significantly lower than that in other groups (P < 0.05). CONCLUSIONS: The methylation of 6 and 42 CG sites may be related to LCHAD gene expression in placenta of L-NAME and ß-2GPI induced preeclampsia-like models respectively; LCHAD gene expression and DNA methylation may not have obvious correlation in LPS and ApoC3 induced preeclampsia-like models. Differences exist in LCHAD DNA methylation in preeclampsia-like models generated by different ways, revealed a molecular basis to expand our understanding of the multi-factorial pathogenesis of preeclampsia.

[The changes of LCHAD in preeclampsia with different clinical features and the correlation with NADPH P47-phox, p38MAPK-α, COX-2 and serum FFA and TG].

OBJECTIVE: To investigate the changes of fatty acid oxidase in the placenta of preeclampsia cases with different clinical features, and the relationship with oxidative stress and inflammatory response. To study the correlation of serum free fatty acid (FFA) and triglycerides (TG) level in early second trimester with the molecular changes of the long-chain fatty acid oxidase in the third trimester. METHODS: This was prospective cohort study, in which cases with singleton pregnancies who archived in Haidian Maternal and Children's Hospital, Beijing, from January 1st 2012 to May 31st, with regular prenatal care were included. Doppler ultrasound was used for screening for the presence of early diastolic notch of uterine artery at 22-24 weeks of gestation. All the 101 cases with the early diastolic notch of uterine artery were included as the notch group, and 377 cases without the early diastolic notch of uterine artery were included as the non-notch group. The perinatal outcomes and the incidence of hypertensive disorders in pregnancy of the two groups were observed. The serum level of FFA and TG was tested, and the mRNA and protein expression of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), P47-phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, p38 mitogen-activated protein kinase a (p38MAPK-α) and cyclooxygenase-2 (COX-2) were detected using real-time quantitative PCR and western blot. The relationship between serum level of FFA and TG and the mRNA and protein expression of LCHAD, NADPH P47-phox, p38MAPK-α and COX-2 of the placental tissue specimens were analyzed. RESULTS: (1) In the notch group, there were 9 cases of early-onset preeclampsia, 15 cases of late-onset preeclampsia and 10 cases of gestational hypertension;and there were 8 cases of late-onset preeclampsia and 18 cases of gestational hypertension in the non-notch group. 15 cases with normal blood pressure in each group were randomly selected as the control group. (2)The serum level of TG of cases of early-onset preeclampsia, late-onset preeclampsia and gestational hypertension in the notch group were (2.0 ± 0.8), (1.8 ± 0.6)and (1.9 ± 0.7)mmol/L, and that of FFA were(0.68 ± 0.26), (0.52 ± 0.10) and (0.52 ± 0.17)mmol/L, respectively. The serum level of TG of cases of late-onset preeclampsia and gestational hypertension in the non-notch group were (1.6 ± 0.6) and (1.4 ± 0.4)mmol/L, and that of FFA were (0.49 ± 0.11) and (0.48 ± 0.05)mmol/L, respectively. The serum level of TG and FFA in the control group were (1.4 ± 0.5) and (0.52 ± 0.06)mmol/L, respectively. The TG level of the notch group was higher than that of the control group, and the difference was statistically significant (P < 0.05). The FFA level of the early-onset preeclampsia in the notch group was higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group, and the difference was statistically significant (P < 0.05). (3) The mRNA expression of LCHAD in the placenta of early-onset preeclampsia in the notch group was significantly lower than that of the late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group (P < 0.01). The mRNA expression of NADPH P47-phox of the early-onset preeclampsia in the notch group were significantly higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group (P < 0.01). The mRNA expression of p38MAPK-α of the early-onset preeclampsia in the notch group were significantly higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group (P < 0.01). The mRNA expression of COX-2 of the early-onset preeclampsia in the notch group were significantly higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and the control group (P < 0.01). (4)The protein expression of LCHAD in the placenta of early-onset preeclampsia in the notch group, late-onset preeclampsia in the notch group and gestational hypertension in the notch group were significantly lower than that of the control group (P < 0.01); and the protein expression of LCHAD in the placenta of early-onset preeclampsia in the notch group was significantly lower than that of late-onset preeclampsia in the non-notch group (P < 0.01). The protein expression of NADPH P47-phox in the placenta of early-onset preeclampsia in the notch group was significantly higher than that of late-onset preeclampsia in the non-notch group and control group (P < 0.05). The protein expression of p38MAPK-α in the placenta of early-onset preeclampsia in the notch group was significantly higher than that of late-onset preeclampsia in the notch group, late-onset preeclampsia in the non-notch group and control group (P < 0.01). The protein expression of COX-2 in the placenta of early-onset preeclampsia in the notch group, late-onset preeclampsia in the notch group, gestational hypertension in the notch group, late-onset preeclampsia in the non-notch group, and gestational hypertension in the non-notch group, were significantly higher than that of control group (P < 0.01). (5)The blood concentration of maternal FFA in the early-onset preeclampsia in the notch group was significantly negatively correlated with the mRNA and protein expression of placental LCHAD (r = -0.810, -0.932, P < 0.01). There was no correlation between maternal TG level and the mRNA and protein expression of placental LCHAD in each group(P > 0.05). (6)The mRNA expression of placental LCHAD in the early-onset preeclampsia in the notch group was significantly negatively correlated with the mRNA expression of placental NADPH P47-phox and COX-2 (r = - 0.877, -0.762, P < 0.05). The mRNA expression of placental LCHAD in the control group was significantly negatively correlated with the mRNA expression of placental COX-2 (r = -0.565, P < 0.01). The protein expression of placental LCHAD in the early-onset preeclampsia in the notch group was significantly negatively correlated with the protein expression of NADPH P47-phox (r = -0.818, P < 0.01). The protein expression of placental LCHAD in the control group was significantly negatively correlated with the protein expression of COX-2 (r = -0.502, P < 0.01). CONCLUSIONS: The placental mRNA and protein expression of long-chain fatty acid oxidation enzymes were different in different clinical features of preeclampsia, which were reduced more obviously in the early-onset preeclampsia in the notch group than that of the late-onset preeclampsia in the notch group, and were negatively correlated with the elevated serum FFA level, significantly enhanced oxidative stress and inflammatory response, but with no correlation with serum TG level.

Docosahexaenoic acid mediates peroxisomal elongation, a prerequisite for peroxisome division.

Peroxisome division is regulated by several factors, termed fission factors, as well as the conditions of the cellular environment. Over the past decade, the idea of metabolic control of peroxisomal morphogenesis has been postulated, but remains largely undefined to date. In the current study, docosahexaenoic acid (DHA, C22:6n-3) was identified as an inducer of peroxisome division. In fibroblasts isolated from patients that carry defects in peroxisomal fatty acid ß-oxidation, peroxisomes are much less abundant than normal cells. Treatment of these patient fibroblasts with DHA induced the proliferation of peroxisomes to the level seen in normal fibroblasts. DHA-induced peroxisomal proliferation was abrogated by treatment with a small inhibitory RNA (siRNA) targeting dynamin-like protein 1 and with dynasore, an inhibitor of dynamin-like protein 1, which suggested that DHA stimulates peroxisome division. DHA augmented the hyper-oligomerization of Pex11pß and the formation of Pex11pß-enriched regions on elongated peroxisomes. Time-lapse imaging analysis of peroxisomal morphogenesis revealed a sequence of steps involved in peroxisome division, including elongation in one direction followed by peroxisomal fission. DHA enhanced peroxisomal division in a microtubule-independent manner. These results suggest that DHA is a crucial signal for peroxisomal elongation, a prerequisite for subsequent fission and peroxisome division.

Systematic review and meta-analysis to estimate the birth prevalence of five inherited metabolic diseases.

Many newborn screening programmes now use tandem mass spectrometry in order to screen for a variety of diseases. However, countries have embraced this technology with a differing pace of change and for different conditions. This has been facilitated by the ability of this diagnostic method to limit analysis to specific metabolites of interest, enabling targeted screening for particular conditions. MS/MS was introduced in 2009 in England to implement newborn bloodspot screening for medium chain acyl-CoA dehydrogenase deficiency (MCADD) raising the possibility of screening for other inherited metabolic disorders. Recently, a pilot screening programme was conducted in order to evaluate the health and economic consequences of screening for five additional inherited metabolic disorders in England. As part of this study we conducted a systematic review and meta-analysis to estimate the birth prevalence of these conditions: maple syrup urine disease, homocystinuria (pyridoxine unresponsive), glutaric aciduria type I, isovaleric acidaemia and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency including trifunctional protein deficiency. We identified a total of 99 studies that were able to provide information on the prevalence of one or more of the disorders. The vast majority of studies were of screening programmes with some reporting on clinically detected cases.

Muscle MRI in patients with long-chain fatty acid oxidation disorders.

INTRODUCTION: Muscle magnetic resonance imaging (MRI) is a useful tool for visualizing abnormalities in neuromuscular disorders. The value of muscle MRI has not been studied in long-chain fatty acid oxidation (lcFAO) disorders. LcFAO disorders may present with metabolic myopathy including episodic rhabdomyolysis. OBJECTIVE: To investigate whether lcFAO disorders are associated with muscle MRI abnormalities. METHODS: Lower body MRI was performed in 20 patients with lcFAO disorders, i.e. three carnitine palmitoyltransferase 2 deficiency (CPT2D), 12 very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), three mitochondrial trifunctional protein deficiency (MTPD) and two isolated long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD). RESULTS: At the time of MRI, four patients had muscle weakness, 14 had muscle pain and 13 were exercise intolerant. Median creatine kinase (CK) level of patients at the day of MRI was 398 U/L (range 35-12,483). T1W and STIR signal intensity (SI) were markedly increased in MTPD patients from girdle to lower leg. VLCADD patients showed predominantly proximal T1W SI changes, whereas LCHADD patients mostly showed distal T1W SI changes. Prominent STIR weighted signal intensity increases of almost all muscle groups were observed in patients with VLCADD and LCHADD with very high CK (>11.000) levels. CONCLUSIONS AND RELEVANCE: lcFAO disorders are associated with specific patterns of increased T1W and STIR signal intensity. These patterns may reflect lipid accumulation and inflammation secondary to lcFAO defects and progressive muscle damage. Future studies are needed to investigate whether muscle MRI might be a useful tool to monitor disease course and to study pathogenesis of lcFAO related myopathy.

Transient central diabetes insipidus induced by ketamine infusion.

OBJECTIVE: Report a case of central diabetes insipidus (DI) associated with ketamine infusion. CASE SUMMARY: A 2-year-old girl with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and stable hypertrophic cardiomyopathy was admitted to the pediatric intensive care with pneumonia. She subsequently developed respiratory failure and required intubation. Continuous ketamine infusion was used for the sedation and facilitation of mechanical ventilation. Shortly after infusion of ketamine, the patient developed DI and responded appropriately to vasopressin. DISCUSSION: The Naranjo adverse drug reaction probability scale indicated a probable relationship between the development of central DI and ketamine. The most likely mechanism involves ketamine's antagonist action on N-methyl-d-aspartate receptors, resulting in inhibition of glutamate-stimulated arginine vasopressin release from the neurohypophysis. CONCLUSION: This is the second case report of ketamine-induced central DI and the only report in children. Clinicians who sedate children with continuous ketamine infusions should monitor patients for developing signs and symptoms of DI by measuring serum sodium and urine output prior to, during, and after ketamine infusion in order to make a timely diagnosis of this potentially serious complication.

Long-chain 3-hydroxy fatty acids accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial trifunctional protein deficiencies uncouple oxidative phosphorylation in heart mitochondria.

Cardiomyopathy is a common clinical feature of some inherited disorders of mitochondrial fatty acid ß-oxidation including mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies. Since individuals affected by these disorders present tissue accumulation of various fatty acids, including long-chain 3-hydroxy fatty acids, in the present study we investigated the effect of 3-hydroxydecanoic (3 HDCA), 3-hydroxydodecanoic (3 HDDA), 3-hydroxytetradecanoic (3 HTA) and 3-hydroxypalmitic (3 HPA) acids on mitochondrial oxidative metabolism, estimated by oximetry, NAD(P)H content, hydrogen peroxide production, membrane potential (ΔΨ) and swelling in rat heart mitochondrial preparations. We observed that 3 HTA and 3 HPA increased resting respiration and diminished the respiratory control and ADP/O ratios using glutamate/malate or succinate as substrates. Furthermore, 3 HDDA, 3 HTA and 3 HPA decreased ΔΨ, the matrix NAD(P)H pool and hydrogen peroxide production. These data indicate that these fatty acids behave as uncouplers of oxidative phosphorylation. We also verified that 3 HTA-induced uncoupling-effect was not mediated by the adenine nucleotide translocator and that this fatty acid induced the mitochondrial permeability transition pore opening in calcium-loaded organelles since cyclosporin A prevented the reduction of mitochondrial ΔΨ and swelling provoked by 3 HTA. The present data indicate that major 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies behave as strong uncouplers of oxidative phosphorylation potentially impairing heart energy homeostasis.

When the usual symptoms become an unusual diagnosis: a case report of trifunctional protein complex.

Disorders of mitochondrial fatty acid b-oxidation should be considered in any infant who presents with unexplained hypoglycemia and/or myopathy. Although disorders of trifunctional protein (TFP) complex including long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial TFP deficiencies are extremely rare, the combined incidence of mitochondrial fatty acid disorders is quite frequent. With the expansion of newborn screening, what were once considered uncommon disorders are being identified with increasing frequency in asymptomatic infants. The following case scenario presents an infant who developed symptoms prior to the completion of newborn screening. This fairly routine course for a late-preterm infant reveals an extremely rare inborn error of metabolism, LCHAD deficiency. An overview of TFP complex, the differential diagnoses as the case unfolds, diagnostic test results, acute care management, and short-term patient follow-up is presented. With experience, health care providers often become accustomed to and expect to see common things regularly. This case presents a scenario which, as it unfolds, appears to be quite common. It turns out, however, to be very uncommon.

Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids.

L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid ß-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor α (PPARα) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadh's expression by PPARα, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ω-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting.

Clinical applications of 3-hydroxy fatty acid analysis by gas chromatography-mass spectrometry.

BACKGROUND: L-3-Hydroxy fatty acids are unusual metabolites and rarely occur in significant quantities in normal human physiology. Genetic defects of both long-chain and medium-/short-chain mitochondrial L-3 hydroxyacyl coenzyme A dehydrogenases (LCHAD, M/SCHAD) have been identified as significant metabolic diseases in humans often with severe clinical phenotypes and pathophysiology that appears to differ from other defects of straight chain fatty acid oxidation. It is felt that accumulation of these atypical fatty acid species may play a role in this pathology. We have therefore developed an assay to measure these compounds in body fluids, and tissue culture medium to help in the diagnosis of these disorders and to better study the effects of 3-hydroxy fatty acid accumulation. METHODS: We have developed a stable isotope dilution, selected ion-monitoring gas chromatography-mass spectrometric assay for the measurement of all 3-hydroxy fatty acids from chain lengths C6 to C18 using 1,2 (13)C-labeled internal standards for all species. Authentic patient samples were utilized to develop reference intervals for control subjects, for those associated with patient samples confirmed at the molecular level to have either LCHAD or M/SCHAD deficiency and for patients who did not have disease but were fasting or on diets high in medium-chain fatty acids. Likewise, skin fibroblasts were obtained from patients with confirmed disease for additional study. Samples were also obtained from the hadh (M/SCHAD) knockout mouse. RESULTS: The measurement of 3-hydroxy fatty acids in patient plasma is a valuable tool in the identification of defects of both enzymes. Severe starvation, prolonged fasting and increased medium-chain triglycerides in the diet produce a profile that is similar to that seen in M/SCHAD deficiency, making this a more difficult condition to diagnose but these biomarkers provide an important clue to the diagnosis, particularly in non-fasted, diet-controlled patients. Fibroblast studies in LCHAD deficiency demonstrate that long-chain 3-hydroxy fatty acid accumulation can be observed in cultured tissues. Incubation of cultured fibroblasts from LCHAD deficient patients with labeled fatty acids demonstrated a process of chain lengthening that has not previously been recognized. CONCLUSIONS: The measurement of body fluid and cultured cell 3-hydroxy fatty acids provides both diagnostic and pathogenic information regarding these genetic diseases of fatty acid oxidation in the mitochondrion. Presently, the measurement of medium- and short-chain species provides a major metabolic biomarker for the recognition of M/SCHAD deficiency.

Diagnosis of a patient with a kinetic variant of medium and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency by newborn screening.

Medium and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is a rare cause of impaired mitochondrial fatty acid oxidation. We present a case report of a patient with hyperinsulinism and homozygosity for a novel mutation causing a kinetic variant of the enzyme. The diagnosis was initially inferred by abnormal newborn screening acylcarnitine analysis with elevated C4-hydroxyacylcarnitine.

Variability in the clinical management of fatty acid oxidation disorders: results of a survey of Canadian metabolic physicians.

INTRODUCTION: There is little robust empirical evidence on which to base treatment recommendations for fatty acid oxidation disorders. While consensus guidelines are important, understanding areas where there is a lack of consensus is also critical to inform priorities for future evaluative research. METHODS: We surveyed Canadian metabolic physicians on the treatment of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, and mitochondrial trifunctional protein (MTP) deficiency. We ascertained physicians' opinions on the use of different interventions for the long-term management of patients as well as for the management of acute illness, focusing on identifying interventions characterized by high variability in opinions. We also investigated factors influencing treatment decisions. RESULTS: We received 18 responses (response rate 45%). Participants focused on avoidance of fasting and increased meal frequency as interventions for the management of MCAD deficiency. For the long-chain disorders, avoidance of fasting remained the most consistently endorsed intervention, with additional highly endorsed treatments differing for VLCAD versus LCHAD/MTP deficiency. L-carnitine supplementation and restriction of dietary fat were characterized by high variability in physicians' opinions, as were several interventions specific to long-chain disorders. Social factors and patient characteristics were important influences on treatment decisions. CONCLUSIONS: Based on our findings we suggest that high priority treatments for rigorous effectiveness studies could include L-carnitine supplementation (MCAD and LCHAD/MTP deficiencies), restriction of dietary fat, and, for the long-chain disorders, feeding practices for breastfed infants and the use of various supplements (essential fatty acids, carbohydrates, cornstarch, multivitamins).

Refined staging for chorioretinopathy in long-chain 3-hydroxyacyl coenzyme A dehydrogenase deficiency.

OBJECTIVE: Neonatal screening and earlier diagnosis have improved the prognosis of long-chain 3-hydroxyacyl coenzyme A dehydrogenase (LCHAD) deficiency, which causes a need to refine the staging of the pigmentary chorioretinopathy and thus improve monitoring and comparability of patients under dietary therapy. METHODS: Seven children with LCHAD deficiency caused by homozygous G1528C mutation attended sequential fundus photography for stage 2 chorioretinopathy in 1997­2006. After arranging 21 pairs of fund us photographs according to the severity of the fundus changes,the images best representing 3 different grades of pigmentary deposits (P1­P3) and retinal pigment epithelial(RPE) atrophy (A1­A3) were chosen as reference photographs.To evaluate the substaging, 29 pairs of photographs were graded according to the reference photographs. RESULTS: In the assessment of pigmentary deposits, the 3 ophthalmologists agreed in 41% and differed by a single substage in 45% of instances (combined weighted ĸ statistic was 0.38, indicating moderate agreement). In pairwise comparisons,the weighted ĸ statistic ranged from 0.31 to 0.56 (agreement, 71­81%). In the assessment of RPE atrophy, all 3 raters agreed in 17% and 2 raters in 70% of instances (combined ĸ statistic 0.018, indicating poor agreement). DISCUSSION: Despite variation in imaging techniques and limitations in the visual assessment of fundus photographs, the agreement obtained in grading the pigmentary deposits was comparable to that reported for photographic grading of retinopathy of prematurity. We recommend photographic documentation and substaging based on reference photographs in the follow-up of LCHAD retinopathy. The refined staging allows a more detailed assessment on the progression of the retinopathy and optimization of the therapeutic protocols in individual patients and between centres using different therapeutic protocols.

Clinical and image-guided chorioretinal findings in long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency.

The purpose of this study is to report clinical, optical coherence tomography (OCT), and fluorescein angiogram/indocyanine green angiography (FA/ICG) findings in patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) enzyme deficiency in two siblings. A 13-year-old girl and her 14-year-old brother presented with progressive decrease in central vision. Clinically, there were blond-looking fundi, diffuse retinal pigment epithelial (RPE) disruption/atrophy in the macula and peripheral retina with choriocapillaris atrophy in both of them. OCT showed RPE irregularity and diffuse disruption of the RPE layer. FA/ICG imaging demonstrated transmitted choroidal fluorescence secondary to diffuse RPE atrophy with no evidence of leakage. Electroretinogram and electrooculogram findings were suggestive of primary abnormality of pigment epithelium. The boy died of cardiac/respiratory illness, whereas his sister is alive at the last follow-up. Abnormal chorioretinal findings in LCHAD patients should be carefully followed. Regular follow-up is recommended to monitor the ocular and systemic status.