GSTZ1-1 Deficiency Activates NRF2/IGF1R Axis in HCC via Accumulation of Oncometabolite Succinylacetone.

The IGF1R signaling is important in the malignant progression of cancer. However, overexpression of IGF1R has not been properly assessed in HCC. Here, we revealed that GSTZ1-1, the enzyme in phenylalanine/tyrosine catabolism, is downregulated in HCC, and its expression was negatively correlated with IGF1R. Mechanistically, GSTZ1-1 deficiency led to succinylacetone accumulation, alkylation modification of KEAP1, and NRF2 activation, thus promoting IGF1R transcription by recruiting SP1 to its promoter. Moreover, inhibition of IGF1R or NRF2 significantly inhibited tumor-promoting effects of GSTZ1 knockout in vivo. These findings establish succinylacetone as an oncometabolite, and GSTZ1-1 as an important tumor suppressor by inhibiting NRF2/IGF1R axis in HCC. Targeting NRF2 or IGF1R may be a promising treatment approach for this subset HCC.

Different Reactivity of Raw Starch from Diverse Potato Genotypes.

Potato starch is one of the most important renewable sources for industrial manufacturing of organic compounds. Currently, it is produced from mixed potato varieties that often are harvested from different fields. Meanwhile, tuber starches of various potato breeds differ in their crystallinity, granule morphology, and other physical and chemical parameters. We studied the reactions of raw potato starches of different origins to chemical and biochemical reactions typically used for industrial starch modification. The results clearly demonstrate that there is a significant difference in the reactivity of the starches of different potato genotypes. While the main products of the transformations are the same, their preparative yields differ significantly. Thus, tuber starch of certain potato varieties may be more suitable for specific industrial purposes. Starch reactivity may potentially be a phenotypical trait for potato breeding to obtain potato starches for various industrial applications.

Loss of fumarylacetoacetate hydrolase causes light-dependent increases in protochlorophyllide and cell death in Arabidopsis.

Fumarylacetoacetate hydrolase (FAH) catalyses the final step of the tyrosine degradation pathway, which is essential to animals but was of unknown importance in plants until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short-day conditions. The sscd1 mutant accumulates succinylacetone (SUAC), an abnormal metabolite caused by loss of FAH. Succinylacetone is an inhibitor of δ-aminolevulinic acid (ALA) dehydratase (ALAD), which is involved in chlorophyll (Chl) biosynthesis. In this study, we investigated whether sscd1 cell death is mediated by Chl biosynthesis and found that ALAD activity is repressed in sscd1 and that protochlorophyllide (Pchlide), an intermediate of Chl biosynthesis, accumulates at lower levels in etiolated sscd1 seedlings. However, it was interesting that Pchlide in sscd1 might increase after transfer from light to dark and that HEMA1 and CHLH are upregulated in the light-dark transition before Pchlide levels increased. Upon re-illumination after Pchlide levels had increased, reactive oxygen species marker genes, including singlet oxygen-induced genes, are upregulated, and the sscd1 cell death phenotype appears. In addition, Arabidopsis WT seedlings treated with SUAC mimic sscd1 in decline of ALAD activity and accumulation of Pchlide as well as cell death. These results demonstrate that increase in Pchlide causes cell death in sscd1 upon re-illumination and suggest that a decline in the Pchlide pool due to inhibition of ALAD activity by SUAC impairs the repression of ALA synthesis from the light-dark transition by feedback control, resulting in activation of the Chl biosynthesis pathway and accumulation of Pchlide in the dark.

Impact of in-feed sodium butyrate or sodium heptanoate protected with medium-chain fatty acids on gut health in weaned piglets challenged with Escherichia coli F4.

Short and medium-chain fatty acids (SCFA and MCFA, respectively) are commonly used as feed additives in piglets to promote health and prevent post-weaning diarrhoea. Considering that the mechanism and site of action of these fatty acids can differ, a combined supplementation could result in a synergistic action. Considering this, it was aimed to assess the potential of two new in-feed additives based on butyrate or heptanoate, protected with sodium salts of MCFA from coconut distillates, against enterotoxigenic Escherichia coli (ETEC) F4+ using an experimental disease model. Two independent trials were performed in 48 early-weaned piglets fed a control diet (CTR) or a diet supplemented with MCFA-protected sodium butyrate (BUT+; Trial 1) or sodium heptanoate (HPT+; Trial 2). After 1 week of adaptation, piglets were challenged with a single oral inoculum of ETEC F4+ (minimum 1.4 · 109 cfu). One animal per pen was euthanised on days 4 and 8 post-inoculation (PI) and the following variables assessed: growth performance, clinical signs, gut fermentation, intestinal morphology, inflammatory mediators, pathogen excretion and colon microbiota. None of the additives recovered growth performance or reduced diarrhoea when compared to the respective negative controls. However, both elicited different responses against ETEC F4+. The BUT+ additive did not lead to reduce E. coli F4 colonisation but enterobacterial counts and goblet cell numbers in the ileum were increased on day 8 PI and this followed higher serum TNF-α concentrations on day 4 PI. The Firmicutes:Bacteroidetes ratio was nevertheless increased. Findings in the HPT+ treatment trial included fewer animals featuring E. coli F4 in the colon and reduced Enterobacteriaceae (determined by 16S RNA sequencing) on day 4 PI. In addition, while goblet cell numbers were lower on day 8 PI, total SCFA levels were reduced in the colon. Results indicate the efficacy of MCFA-protected heptanoate against ETEC F4+ and emphasise the potential trophic effect of MCFA-protected butyrate on the intestinal epithelium likely reinforcing the gut barrier.

Multi-Omics Approach for Studying Tears in Treatment-Naïve Glaucoma Patients.

Primary open-angle glaucoma (POAG) represents the leading cause of irreversible blindness worldwide and is a multifactorial, chronic neurodegenerative disease characterized by retinal ganglion cell and visual field loss. There are many factors that are associated with the risk of developing POAG, with increased intraocular pressure being one of the most prevalent. Due to the asymptomatic nature of the disease, the diagnosis of POAG often occurs too late, which necessitates development of new effective screening strategies for early diagnosis of the disease. However, this task still remains unfulfilled. In order to provide further insights into the pathophysiology of POAG, we applied a targeted metabolomics strategy based on a high-throughput screening method for the determination of tear amino acids, free carnitine, acylcarnitines, succinylacetone, nucleosides, and lysophospholipids in naïve to therapy glaucomatous patients and normal controls. Also, we conducted proteomic analyses of the whole lacrimal fluid and purified extracellular vesicles obtained from POAG patients and healthy subjects. This multi-omics approach allowed us to conclude that POAG patients had lower levels of certain tear amino acids and lysophospholipids compared with controls. These targeted analyses also highlighted the low amount of acetylcarnitine (C2) in POAG patient which correlated well with proteomics data. Moreover, POAG tear proteins seemed to derive from extracellular vesicles, which carried a specific pro-inflammatory protein cargo.

Response of gastrointestinal fermentative activity and colonic microbiota to protected sodium butyrate and protected sodium heptanoate in weaned piglets challenged with ETEC F4.

This study aimed to evaluate the potential of two new fat-protected butyrate or heptanoate salts to improve gut health and control post-weaning colibacillosis in weaning piglets challenged with enterotoxigenic Escherichia coli (ETEC) F4+, particularly focusing on their impact on intestinal microbiota and fermentative activity along the gastrointestinal tract (GIT). Seventy-two 21-d-old pigs were fed a plain diet (CTR) or supplemented with sodium butyrate (BUT) or sodium heptanoate (HPT), both at 0.3%. After a week of adaptation, animals were orally challenged at days 8 and 9 with 5.8 · 109 and 6.6 · 1010 cfu, respectively, and were euthanised on d 4 and d 8 post-inoculation (PI) (n = 8) to collect blood, digesta and tissue samples and characterise microbial groups, pathogen loads (qPCR), fermentation, ileal histomorphometry and immune markers. Colonic microbiota was analysed by 16S rRNA gene MiSeq sequencing. Supplementing both acid salts did not compensate clinical challenge effects nor performance impairments and neither histomorphometry nor serum biomarkers. Changes in the gastric fermentative activity were registered, BUT reducing lactic acid concentrations (day 8 PI), and with HPT fewer animals presenting detectable concentrations of propionic, butyric and valeric acids. At ileum BUT increased acetic acid concentration (day 8 PI), and both additives reduced short-chain fatty acids (SCFA) in the colon. Increases in enterobacteria and coliforms counts in ileal digesta (day 4 PI, p < 0.10) and mucosa scrapes (p < 0.05) were registered although E. coli F4 gene copies were unaffected. Regarding changes in the colonic microbiota (day 4 PI), Prevotellaceae and Prevotella were promoted with BUT supplementation whereas only minor groups were modified in HPT-treated animals. Summarising, although the pathogen loads or inflammatory mediators remained unresponsive, butyrate and heptanoate showed a significant impact on microbial fermentation along the whole GIT, being able to modify different bacterial groups at the colon. It could be hypothesised that these effects might be mediated by a carry-over effect of the changes observed in gastric fermentation, but possibly also to a better nutrient digestion in the foregut as a result of the reduced colonic SCFA concentrations.

Chronic Phenotype Characterization of a Large-Animal Model of Hereditary Tyrosinemia Type 1.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by deficiency in fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolic pathway. In this study, we investigated whether fumarylacetoacetate hydrolase deficient (FAH-/-) pigs, a novel large-animal model of HT1, develop fibrosis and cirrhosis characteristic of the human disease. FAH-/- pigs were treated with the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3 cyclohexandione (NTBC) at a dose of 1 mg/kg per day initially after birth. After 30 days, they were assigned to one of three groups based on dosing of NTBC. Group 1 received ≥0.2 mg/kg per day, group 2 cycled on/off NTBC (0.05 mg/kg per day × 1 week/0 mg/kg per day × 3 weeks), and group 3 received no NTBC thereafter. Pigs were monitored for features of liver disease. Animals in group 1 continued to have weight gain and biochemical analyses comparable to wild-type pigs. Animals in group 2 had significant cessation of weight gain, abnormal biochemical test results, and various grades of fibrosis and cirrhosis. No evidence of hepatocellular carcinoma was detected. Group 3 animals declined rapidly, with acute liver failure. In conclusion, the FAH-/- pig is a large-animal model of HT1 with clinical characteristics that resemble the human phenotype. Under conditions of low-dose NTBC, FAH-/- pigs developed liver fibrosis and portal hypertension, and thus may serve as a large-animal model of chronic liver disease.

Newborn Screening for Hereditary Tyrosinemia Type I in Québec: Update.

Hereditary tyrosinemia type I (HTI) is a rare autosomal recessive disorder caused by a fumarylacetoacetate hydrolase (FAH) deficiency. If untreated, its acute form is characterized by hepatic failure, renal dysfunction and neurological crisis, and may lead to death. Due to a genetic founder effect in the French-Canadian population, the prevalence of HTI is increased in the province of Quebec (1/19 819), with the IVS12 + 5G>A (1062 + 5G>A) splice site mutation responsible for more than 90% of mutated alleles. Universal newborn screening for (HT1) was thus established in 1970, and close to four million infants have been tested so far, allowing to identify 185 of the 190 affected newborns. During the 1970-1997 period, 2,249,000 newborns were screened at 3-7 days of life on dried filter paper blood spots by tyrosine (Tyr) concentration followed by indirect colorimetric semi-quantitative and quantitative (Q) succinylacetone (SA) testing (red blood cells δ-aminolevulinate dehydratase inhibition), with immunoreactive FAH as the confirmatory test. This approach allowed to identify 118 of 123 affected newborns. In 1998, owing to earlier hospital discharge and increased rate of breastfeeding, four cases were missed within the same year as the discriminating power of blood Tyr became inadequate. Thus, the screening algorithm was modified: indirect semi-quantitative SA measurement became the first-tier test between 1998 and 2014, and direct SA measurement by tandem mass spectrometry (MS/MS) was implemented in 2014, followed by indirect quantitative SA measurement as second tier test. Confirmation is performed by plasmatic amino acid profile and molecular testing. During the 1998-2016 period, more than 1,5 million neonates have been tested (90% sampled between 24 and 48 h of life): 67 of the 67 HTI cases were identified. Both indirect and direct SA measurement as the initial HTI screening test proved to be highly sensitive and specific, with positive and negative predicting value of 79% and 100% respectively.

Hepatorenal Tyrosinemia in Mexico: A Call to Action.

Hepatorenal tyrosinemia is a treatable metabolic disease characterized by progressive liver failure, renal damage and pronounced coagulopathy. Its clinical diagnosis is difficult because of its low prevalence and heterogeneous symptoms. In developed countries, expanded newborn screening, based on succinylacetone quantification by tandem mass spectrometry, has been very valuable in the early detection of hepatorenal tyrosinemia, providing the opportunity for rapid treatment of affected patients. In developing countries without systematic expanded newborn screening, however, diagnosis and treatment of this disease remain major challenges, as genetic diseases in these countries are not a health priority and there are few referral centers for infants with inherited errors of metabolism. This chapter describes the diagnosis, follow-up and outcome of 20 Mexican patients with hepatorenal tyrosinemia. This chapter also constitutes a call to action to pediatricians, gastroenterologists, geneticists and other health professionals, and to academic organizations, health authorities and patient advocacy groups, to promote early patient detection and treatment, reducing the unacceptably high mortality rate (75%) in Mexican infants with this potentially deadly but eminently treatable condition.

The Québec NTBC Study.

In this chapter we describe the current Quebec NTBC Study protocol. Quebec's unique characteristics have influenced the development of the protocol, including a high prevalence of hepatorenal tyrosinemia (HT1), universal newborn screening for HT1, availability of treatment with nitisinone (NTBC) and special diet, a large territory, where HT1 treatment is coordinated by a small number of centers. Screened newborns are seen within 3 weeks of birth. Patients with liver dysfunction (prolonged prothrombin time and/or international normalized ratio (INR) provide sensitive, rapidly available indicators) are treated by NTBC and special diet. The specific diagnosis is confirmed by diagnostic testing for succinylacetone (SA) in plasma and urine samples obtained before treatment. After an initial period of frequent surveillance, stable patients are followed every 3 months by assay of plasma amino acids and NTBC and plasma and urine SA. Abdominal ultrasound is done every 6 months. Patients have an annual visit to the coordinating center that includes multidisciplinary evaluations in metabolic genetics, hepatology, imaging (for abdominal ultrasound and magnetic resonance imaging) and other specialties as necessary. If hepatocellular carcinoma is suspected by imaging and/or because of progressive elevation of alphafetoprotein, liver transplantation is discussed. To date, no patient in whom treatment was started before 1 month of age has developed hepatocellular carcinoma, after surveillance for up to 20 years in some. This patient group is the largest in the world that has been treated rapidly following newborn screening. The protocol continues to evolve to adapt to the challenges of long term surveillance.

Diagnosing Hepatorenal Tyrosinaemia in Europe: Newborn Mass Screening Versus Selective Screening.

Hepatorenal tyrosinaemia (HT1) is a serious condition that used to be fatal before the advent of nitisinone (NTBC, Orfadine®) as a therapeutic option. We have recently shown that selective screening is inadequate as initial symptoms are often uncharacteristic which leads to a considerable delay in diagnosis and treatment. This has a negative impact on morbidity and mortality as well as long-term outcome. For example, the odds ratio to develop hepatocellular carcinoma is 12.7 when treatment is initiated after the first birthday compared to start of treatment in the neonatal period. Timely diagnosis is only possible when neonatal mass screening is operational. HT1 meets all the criteria for neonatal mass screening at a clinical and analytical level. The natural course of the disease is well known, clinically there is a latent phase in most patients when presymptomatic treatment can be initiated. There are no mild phenotypes which do not require treatment. Using succinylacetone as the screening parameter a highly specific and sensitive test is available with acceptable financial burden. Neonatal mass screening for HT1 is acceptable to the target population as it can be performed simultaneously with the already existing screening tests in dried blood, there are no false negative and false positive cases and the financial burden to the health system is moderate. An efficient treatment is available with nitisinone and protein-reduced diet supplemented with special amino acid mixtures. Despite compelling evidence in favour of a neonatal mass screening for HT1 only 57% of European centres taking part in our recent cross-sectional study have included HT1 in their newborn screening programme.

Silent Tyrosinemia Type I Without Elevated Tyrosine or Succinylacetone Associated with Liver Cirrhosis and Hepatocellular Carcinoma.

Tyrosinemia type I (TYRSN1, TYR I) is caused by fumarylacetoacetate hydrolase (FAH) deficiency and affects approximately one in 100,000 individuals worldwide. Pathogenic variants in FAH cause TYRSN1, which induces cirrhosis and can progress to hepatocellular carcinoma (HCC). TYRSN1 is characterized by the production of a pathognomonic metabolite, succinylacetone (SUAC) and is included in the Recommended Uniform Screening Panel for newborns. Treatment intervention is effective if initiated within the first month of life. Here, we describe a family with three affected children who developed HCC secondary to idiopathic hepatosplenomegaly and cirrhosis during infancy. Whole exome sequencing revealed a novel homozygous missense variant in FAH (Chr15(GRCh38):g.80162305A>G; NM_000137.2:c.424A > G; NP_000128.1:p.R142G). This novel variant involves the catalytic pocket of the enzyme, but does not result in increased SUAC or tyrosine, making the diagnosis of TYRSN1 problematic. Testing this novel variant using a rapid, in vivo somatic mouse model showed that this variant could not rescue FAH deficiency. In this case of atypical TYRSN1, we show how reliance on SUAC as a primary diagnostic test can be misleading in some patients with this disease. Augmentation of current screening for TYRSN1 with targeted sequencing of FAH is warranted in cases suggestive of the disorder.

Differential effects of octanoate and heptanoate on myocardial metabolism during extracorporeal membrane oxygenation in an infant swine model.

Nutritional energy support during extracorporeal membrane oxygenation (ECMO) should promote successful myocardial adaptation and eventual weaning from the ECMO circuit. Fatty acids (FAs) are a major myocardial energy source, and medium-chain FAs (MCFAs) are easily taken up by cell and mitochondria without membrane transporters. Odd-numbered MCFAs supply carbons to the citric acid cycle (CAC) via anaplerotic propionyl-CoA as well as acetyl-CoA, the predominant ß-oxidation product for even-numbered MCFA. Theoretically, this anaplerotic pathway enhances carbon entry into the CAC, and provides superior energy state and preservation of protein synthesis. We tested this hypothesis in an immature swine model undergoing ECMO. Fifteen male Yorkshire pigs (26-45 days old) with 8-h ECMO received either normal saline, heptanoate (odd-numbered MCFA), or octanoate (even-numbered MCFA) at 2.3 µmol·kg body wt(-1)·min(-1) as MCFAs systemically during ECMO (n = 5/group). The 13-carbon ((13)C)-labeled substrates ([2-(13)C]lactate, [5,6,7-(13)C3]heptanoate, and [U-(13)C6]leucine) were systemically infused as metabolic markers for the final 60 min before left ventricular tissue extraction. Extracted tissues were analyzed for the (13)C-labeled and absolute concentrations of metabolites by nuclear magnetic resonance and gas chromatography-mass spectrometry. Octanoate produced markedly higher myocardial citrate concentration, and led to a higher [ATP]-to-[ADP] ratio compared with other groups. Unexpectedly, octanoate and heptanoate increased the flux of propionyl-CoA relative to acetyl-CoA into the CAC compared with control. MCFAs promoted increases in leucine oxidation, but were not associated with a difference in protein synthesis rate. In conclusion, octanoate provides energetic advantages to the heart over heptanoate.

Tyrosinemia type I: clinical and biochemical analysis of patients in Mexico.

INTRODUCTION: Hepatorenal tyrosinemia (HT1) is a treatable, inherited, metabolic disease characterized by progressive liver failure with pronounced coagulopathy. The aim of this study is to describe the clinical, biochemical, and histopathological findings in a group of Mexican HT1 patients and their outcome. MATERIAL AND METHODS: Medical records of HT1 patients diagnosed between 1995 and 2011 were analyzed. The diagnosis of HT1 was confirmed by detection of succinylacetone in urine or blood. RESULTS: Sixteen nonrelated HT1 cases were analyzed. Mean age at clinical onset was 9 months, and the mean age at diagnosis was 16.3 months. Main clinical findings were hepatomegaly, splenomegaly, cirrhosis, liver failure, tubulopathy, nephromegaly, Fanconi syndrome, seizures and failure to thrive. Histopathological findings were cirrhosis, fibrosis and steatosis. The HT1 group had a mortality rate of 78%. Patients who received supportive care or nutritional treatment had a 3-year survival rate of 10%. For those who underwent liver transplantation, the 6-year survival rate was 60%. In most cases pharmacological treatment with nitisinone and special dietary products were not available. The leading causes of death were fulminant liver failure, metastatic hepatocellular carcinoma, and porphyria-like neurologic crisis. Newborn screening programs in combination with the availability of orphan drugs, proper monitoring, genetic counseling, and clinical practice guidelines are needed to enable physicians to identify the disease, delay its progression, and improve patients' quality of life. CONCLUSION: The devastating natural history of HT1 is still observed in Mexican patients because they are not diagnosed and treated during the early stages of the disease.

Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I.

Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH-/- mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.

Odd- and even-numbered medium-chained fatty acids protect against glutathione depletion in very long-chain acyl-CoA dehydrogenase deficiency.

Recent trials have reported the ability of triheptanoin to improve clinical outcomes for the severe symptoms associated with long-chain fatty acid oxidation disorders, including very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. However, the milder myopathic symptoms are still challenging to treat satisfactorily. Myopathic pathogenesis is multifactorial, but oxidative stress is an important component. We have previously shown that metabolic stress increases the oxidative burden in VLCAD-deficient cell lines and can deplete the antioxidant glutathione (GSH). We investigated whether medium-chain fatty acids provide protection against GSH depletion during metabolic stress in VLCAD-deficient fibroblasts. To investigate the effect of differences in anaplerotic capacity, we included both even-(octanoate) and odd-numbered (heptanoate) medium-chain fatty acids. Overall, we show that modulation of the concentration of medium-chain fatty acids in culture media affects levels of GSH retained during metabolic stress in VLCAD-deficient cell lines but not in controls. Lowered glutamine concentration in the culture media during metabolic stress led to GSH depletion and decreased viability in VLCAD deficient cells, which could be rescued by both heptanoate and octanoate in a dose-dependent manner. Unlike GSH levels, the levels of total thiols increased after metabolic stress exposure, the size of this increase was not affected by differences in cell culture medium concentrations of glutamine, heptanoate or octanoate. Addition of a PPAR agonist further exacerbated stress-related GSH-depletion and viability loss, requiring higher concentrations of fatty acids to restore GSH levels and cell viability. Both odd- and even-numbered medium-chain fatty acids efficiently protect VLCADdeficient cells against metabolic stress-induced antioxidant depletion.

The Pharmacokinetics of Triheptanoin and Its Metabolites in Healthy Subjects and Patients With Long-Chain Fatty Acid Oxidation Disorders.

Long-chain fatty acid oxidation disorders (LC-FAODs) are a group of life-threatening autosomal recessive disorders caused by defects in nuclear genes encoding mitochondrial enzymes involved in the conversion of dietary long-chain fatty acids into energy. Triheptanoin is an odd-carbon, medium-chain triglyceride consisting of 3 fatty acids with 7 carbons each on a glycerol backbone developed to treat adult and pediatric patients with LC-FAODs. The pharmacokinetics of triheptanoin and circulating metabolites were explored in healthy subjects and patients with LC-FAODs using noncompartmental analyses. Systemic exposure to triheptanoin following an oral administration was negligible, as triheptanoin is extensively hydrolyzed to glycerol and heptanoate in the gastrointestinal tract. Multiple peaks for triheptanoin metabolites were observed in the plasma following oral administration of triheptanoin, generally coinciding with the time that meals were served. Heptanoate, the pharmacologically active metabolite of triheptanoin supplementing energy sources in patients with LC-FAODs, showed the greatest exposure among the metabolites of triheptanoin in human plasma following oral administration of triheptanoin. The exposure of heptanoate was approximately 10-fold greater than that of beta-hydroxypentoate, a downstream metabolite of heptanoate. Exposure to triheptanoin metabolites appeared to increase following multiple doses as compared with the single dose, and with the increase in triheptanoin dose levels.

Interrelations between C4 ketogenesis, C5 ketogenesis, and anaplerosis in the perfused rat liver.

We investigated the interrelations between C(4) ketogenesis (production of beta-hydroxybutyrate + acetoacetate), C(5) ketogenesis (production of beta-hydroxypentanoate + beta-ketopentanoate), and anaplerosis in isolated rat livers perfused with (13)C-labeled octanoate, heptanoate, or propionate. Mass isotopomer analysis of C(4) and C(5) ketone bodies and of related acyl-CoA esters reveal that C(4) and C(5) ketogenesis share the same pool of acetyl-CoA. Although the uptake of octanoate and heptanoate by the liver are similar, the rate of C(5) ketogenesis from heptanoate is much lower than the rate of C(4) ketogenesis from octanoate. This results from the channeling of the propionyl moiety of heptanoate into anaplerosis of the citric acid cycle. C(5) ketogenesis from propionate is virtually nil because acetoacyl-CoA thiolase does not favor the formation of beta-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA. Anaplerosis and gluconeogenesis from heptanoate are inhibited by octanoate. The data have implications for the design of diets for the treatment of long chain fatty acid oxidation disorders, such as the triheptanoin-based diet.

Hereditary tyrosinemia type 1 metabolites impair DNA excision repair pathways.

Hereditary tyrosinemia type 1 is an autosomal recessive metabolic disorder, which is caused by a defective fumarylacetoacetate hydrolase enzyme, and consequently metabolites such as succinylacetone and p-hydroxyphenylpyruvate accumulate. We used a modified comet assay to determine the effect of these metabolites on base- and nucleotide excision repair pathways. Our results indicate that the metabolites affected the repair mechanisms differently, since the metabolites had a bigger detrimental effect on BER than on NER.

Nonactin biosynthesis: unexpected patterns of label incorporation from 4,6-dioxoheptanoate show evidence of a degradation pathway for levulinate through propionate in Streptomyces griseus.

The polyketide nonactin, a polyketide possessing antitumor and antibacterial activity, is produced by an unusual biosynthesis pathway in Streptomyces griseus that uses both enantiomers of the nonactin precursor, nonactic acid. Despite many studies with labeled precursors, much of the biosynthesis pathway remains unconfirmed, particularly the identity of the last achiral intermediate in the pathway, which is believed to be 4,6-diketoheptanoyl-CoA. We set out to confirm the latter hypothesis with feeding studies employing [4,5-(13)C(2)]-, [5,6-(13)C(2)]-, and [6,7-(13)C(2)]-4,6-diketoheptanoate thioester derivatives. In each case the isotopic label was incorporated efficiently into nonactin; however, at positions inconsistent with the currently accepted biosynthesis pathway. To resolve the discrepancy, we conducted additional feeding studies with a [3,4-(13)C(2)]levulinate thioester derivative and again observed efficient label incorporation. The latter result was intriguing, as levulinate is not an obvious precursor to nonactin. Levulinate, however, is known to be efficiently degraded into propionate even though the pathway for the conversion is not known. On the basis of both our levulinate and diketoheptanoate isotope incorporation data we can now postulate a pathway from levulinate to propionate that can also account for the conversion of 4,6-diketoheptanoate into levulinate in S. griseus.