Neonatal screening for isovaleric aciduria: Reducing the increasingly high false-positive rate in Germany.

Newborn screening (NBS) for isovaleric acidemia (IVA) is performed by flow injection tandem mass spectrometry quantifying C5 carnitines (C5). Isovalerylcarnitine, however, is isomeric with pivaloylcarnitine which can be present in blood due to maternal use of pivaloylester-containing antibiotics, available in Germany since late 2016. During a 36-month period (January 19-December 21), all newborns screened in Hamburg with a C5 above cutoff (NeoGram®: 0.50 µmol/L or Neobase®2: 0.45 µmol/L) were included in the study. As a second-tier test, a simple ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to differentiate the C5 isomers pivaloyl-, 2-methylbutyryl-, isovaleryl-, and valerylcarnitine. Out of 156 772 newborns tested, one turned out to have genetically proven IVA while 99 were false positive (C5: 0.5-8.2 µmol/L) due to the presence of pivaloylcarnitine. These cases have increased year by year and show local clusters. Retrospective analysis of another 39 cases from 287 206 neonates tested at the NBS center in Heidelberg with C5 elevation (0.9-10.6 µmol/L) but clinical and biochemical exclusion of IVA yielded evidence of pivaloylcarnitine in all cases. Inclusion of a second-tier test into NBS significantly reduces the high and increasing false-positive rate of IVA screening. This avoids further diagnostic steps, prevents unnecessary stress and anxiety of parents in a remarkably high number of cases. If Hamburg data of 2021 are extrapolated to all of Germany, one can assume around 800 (1‰) false-positive cases in comparison to an average of two classic IVA cases per year. Unless licensing of pivaloylester-containing drugs for use during pregnancy is reconsidered, a second-tier test for C5 determination is indispensable.

GFI1B acts as a metabolic regulator in hematopoiesis and acute myeloid leukemia.

Recent studies highlighted the role of transcription factors in metabolic regulation during hematopoiesis and leukemia development. GFI1B is a transcriptional repressor that plays a critical role in hematopoiesis, and its expression is negatively related to the prognosis of acute myeloid leukemia (AML) patients. We earlier reported a change in the metabolic state of hematopoietic stem cells upon Gfi1b deletion. Here we explored the role of Gfi1b in metabolism reprogramming during hematopoiesis and leukemogenesis. We demonstrated that Gfi1b deletion remarkably activated mitochondrial respiration and altered energy metabolism dependence toward oxidative phosphorylation (OXPHOS). Mitochondrial substrate dependency was shifted from glucose to fatty acids upon Gfi1b deletion via upregulating fatty acid oxidation (FAO). On a molecular level, Gfi1b epigenetically regulated multiple FAO-related genes. Moreover, we observed that metabolic phenotypes evolved as cells progressed from preleukemia to leukemia, and the correlation between Gfi1b expression level and metabolic phenotype was affected by genetic variations in AML cells. FAO or OXPHOS inhibition significantly impeded leukemia progression of Gfi1b-KO MLL/AF9 cells. Finally, we showed that Gfi1b-deficient AML cells were more sensitive to metformin as well as drugs implicated in OXPHOS and FAO inhibition, opening new potential therapeutic strategies.

Bioenergetic dysfunction in a zebrafish model of acute hyperammonemic decompensation.

Acute hyperammonemic encephalopathy is a life-threatening manifestation of individuals with urea cycle disorders, which is associated with high mortality rates and severe neurological sequelae in survivors. Cerebral bioenergetic failure has been proposed as one of the key mechanisms underlying hyperammonemia-induced brain damage, but data supporting this hypothesis remain inconclusive and partially contradictory. Using a previously established zebrafish model of acute hyperammonemic decompensation, we unraveled that acute hyperammonemia leads to a transamination-dependent withdrawal of 2-oxoglutarate (alpha-ketoglutarate) from the tricarboxylic acid (TCA) cycle with consecutive TCA cycle dysfunction, ultimately causing impaired oxidative phosphorylation with ATP shortage, decreased ATP/ADP-ratio and elevated lactate concentrations. Thus, our study supports and extends the hypothesis that cerebral bioenergetic dysfunction is an important pathophysiological hallmark of hyperammonemia-induced neurotoxicity.

Simultaneous Serial Transverse Enteroplasty (STEP) in Size Mismatch Small Bowel Transplantations.

BACKGROUND: Small bowel transplantation (SBTX) in children receiving larger grafts from adults can be challenging because of size mismatch. The aim of the present study was to assess whether a simultaneous serial transverse enteroplasty (STEP) can address the problem of size mismatch. METHODS: Three different size ratio groups between donors and recipients were compared in a porcine model with a 14-day follow-up. The groups were size matched, size mismatched (1:3.8 weight ratio), and size mismatched + STEP (each n = 8). RESULTS: It was technically feasible to simultaneously perform a STEP and SBTX of a mismatched intestinal segment. The postoperative clinical course was uneventful. No signs of bleeding, leakage, stenosis, or ileus were observed and the intestinal segment was well perfused at relaparotomy. Body weight decreased in all groups, but the percentage decrease was lowest in the mismatched + STEP group. Vital enterocyte masses were similar in all the groups (citrulline levels) and the nutritional status was best in the STEP group (transferrin levels, p = 0.04). CONCLUSIONS: We have demonstrated that a simultaneous STEP and SBTX procedure is technically feasible and clinically useful in overcoming the challenges associated with size mismatched SBTX. Our short-term findings justify further investigation in a larger series to elucidate the long-term outcomes of this procedure.

Pharmacologic rescue of hyperammonemia-induced toxicity in zebrafish by inhibition of ornithine aminotransferase.

Hyperammonemia is the common biochemical hallmark of urea cycle disorders, activating neurotoxic pathways. If untreated, affected individuals have a high risk of irreversible brain damage and mortality. Here we show that acute hyperammonemia strongly enhances transamination-dependent formation of osmolytic glutamine and excitatory glutamate, thereby inducing neurotoxicity and death in ammoniotelic zebrafish larvae via synergistically acting overactivation of NMDA receptors and bioenergetic impairment induced by depletion of 2-oxoglutarate. Intriguingly, specific and irreversible inhibition of ornithine aminotransferase (OAT) by 5-fluoromethylornithine rescues zebrafish from lethal concentrations of ammonium acetate and corrects hyperammonemia-induced biochemical alterations. Thus, OAT inhibition is a promising and effective therapeutic approach for preventing neurotoxicity and mortality in acute hyperammonemia.

Maleic Acid--but Not Structurally Related Methylmalonic Acid--Interrupts Energy Metabolism by Impaired Calcium Homeostasis.

Maleic acid (MA) has been shown to induce Fanconi syndrome via disturbance of renal energy homeostasis, though the underlying pathomechanism is still under debate. Our study aimed to examine the pathomechanism underlying maleic acid-induced nephrotoxicity. Methylmalonic acid (MMA) is structurally similar to MA and accumulates in patients affected with methymalonic aciduria, a defect in the degradation of branched-chain amino acids, odd-chain fatty acids and cholesterol, which is associated with the development of tubulointerstitial nephritis resulting in chronic renal failure. We therefore used MMA application as a control experiment in our study and stressed hPTECs with MA and MMA to further validate the specificity of our findings. MMA did not show any toxic effects on proximal tubule cells, whereas maleic acid induced concentration-dependent and time-dependent cell death shown by increased lactate dehydrogenase release as well as ethidium homodimer and calcein acetoxymethyl ester staining. The toxic effect of MA was blocked by administration of single amino acids, in particular L-alanine and L-glutamate. MA application further resulted in severe impairment of cellular energy homeostasis on the level of glycolysis, respiratory chain, and citric acid cycle resulting in ATP depletion. As underlying mechanism we could identify disturbance of calcium homeostasis. MA toxicity was critically dependent on calcium levels in culture medium and blocked by the extra- and intracellular calcium chelators EGTA and BAPTA-AM respectively. Moreover, MA-induced cell death was associated with activation of calcium-dependent calpain proteases. In summary, our study shows a comprehensive pathomechanistic concept for MA-induced dysfunction and damage of human proximal tubule cells.