Propionate Pathway Research Articles

Transcriptomic and proteomic changes associated with cobalamin-dependent propionate production by the rumen bacterium Xylanibacter ruminicola.

In ruminants, the rumen microbial community plays a critical role in nutrition through the fermentation of feed to provide vital energy substrates for the host animal. Propionate is a major rumen fermentation end-product and increasing its production is desirable given its importance in host glucose production and impact on greenhouse gas production. Vitamin B12 (cobalamin) can induce propionate production in the prominent rumen bacterium Xylanibacter ruminicola, but it is not fully understood how cobalamin regulates propionate pathway activity. Contrary to expectation, we found that cobalamin supplementation had little effect on propionate pathway expression at transcriptome and proteome levels, with minor upregulation of genes encoding the cobalamin-dependent enzyme of the pathway. These findings provide new insights into factors that regulate propionate production and suggest that cobalamin-dependent propionate production by X. ruminicola is controlled post-translationally.

Evidence That Long-Term Treatment Prevents Tissue Oxidative Damage in Patients With Inherited Disorders of the Propionate Pathway.

Propionic and methylmalonic acidemias (PAcidemia and MMAcidemia, respectively) are genetic disorders clinically characterized by metabolic decompensation associated with life-threatening encephalopathic episodes in the neonatal period. Adequate and rapid therapeutic management is essential for patients' survival and prognosis. In this study, a restricted protein diet associated with L-carnitine (LC) supplementation was shown to decrease mortality and morbidity in patients affected by these disorders probably by decreasing the accumulation of the major metabolites and therefore their toxicity. Since oxidative stress was proposed as a contributing mechanism of tissue damage in PAcidemia and MMAcidemia and LC has potent antioxidant properties, our objective in this work was to investigate the effects of a long-term therapy consisting of reduced protein intake associated with LC supplementation on oxidative damage markers in patients affected by these diseases. We measured urinary isoprostanes, di-tyrosine, and oxidized guanine species, which reflect oxidative damage to lipids, proteins, and DNA/RNA, respectively, as well as the concentrations of NO products (nitrate plus nitrite) in patients untreated or submitted to short-term or a long-term treatment. Results revealed significant increases of isoprostanes, di-tyrosine, and oxidized guanine species, as well as a moderate nonsignificant increase of NO levels in the untreated patients, relatively to controls. Furthermore, these altered markers were attenuated after short-term treatment and normalized after prolonged treatment. In conclusion, data from this work show for the first time that long-standing treatment of patients with disorders of the propionate pathway can protect against oxidative damage. However, it remains to be elucidated whether oxidative stress identified in this study directly correlates with the clinical conditions of the affected patients.

Examination of the effects of dry storage stress on mitochondrial energy synthesis in the scallop Mizuhopecten yessoensis

Postharvest dry storage was simulated by storing scallops at 4 °C for different durations. The quantification of 40 energy metabolites related to the glycolysis, tricarboxylic acid (TCA) cycle and oxidative phosphorylation pathways in the scallop adductor muscle was performed by LC–MS/MS. The results showed that during the early stage (0–6 h), 6-phosphofructokinase-2 (PFK-2) in glycolysis was inhibited, and the TCA cycle was maintained at a low level. Concurrently, the aspartate pathway and opine pathway were initiated, and alanine and opines were the early metabolites found during this stage. During the middle and late stages (6–12 h) of dry storage, muscle pH decreased. Phosphoenolpyruvate carboxy kinase (PEPCK) was activated, leading to the competitive inhibition of pyruvate synthesis by PEPCK. Phosphoenolpyruvate enters the reverse TCA cycle via the phosphoenolpyruvate → malate pathway to produce succinate. The increase in succinate further induced the succinyl-CoA → propionate pathway, and the end products were acetate and propionate. Alteration of the metabolic pathway was observed, leading to a change in the NADH level. Subsequently, a fluctuating mitochondrial membrane potential was observed. Reimmersion after 6 h of dry storage promoted the recovery of the muscle TCA cycle. Nevertheless, after long-term dry storage (24 h), reimmersion storage did not restore the succinate → oxaloacetate metabolism in the TCA cycle. High-intensity air exposure had a long-term and irreversible effect on mitochondrial energy metabolism. Therefore, it is concluded that the dynamics of mitochondrial metabolism may be sensitive to dry exposure during postharvest operations.

Genomic reconstruction of short-chain fatty acid production by the human gut microbiota.

Short-chain fatty acids (SCFAs) including acetate, formate, propionate, and butyrate are the end products of dietary fiber and host glycan fermentation by the human gut microbiota (HGM). SCFAs produced in the column are of utmost importance for host physiology and health. Butyrate and propionate improve gut health and play a key role in the neuroendocrine and immune systems. Prediction of HGM metabolic potential is important for understanding the influence of diet and HGM-produced metabolites on human health. We conducted a detailed metabolic reconstruction of pathways for the synthesis of SCFAs and L- and D-lactate, as additional fermentation products, in a reference set of 2,856 bacterial genomes representing strains of >800 known HGM species. The reconstructed butyrate and propionate pathways included four and three pathway variants, respectively, that start from different metabolic precursors. Altogether, we identified 48 metabolic enzymes, including five alternative enzymes in propionate pathways, and propagated their occurrences across all studied genomes. We established genomic signatures for reconstructed pathways and classified genomes according to their simplified binary phenotypes encoding the ability (“1”) or inability (“0”) of a given organism to produce SCFAs. The resulting binary phenotypes combined into a binary phenotype matrix were used to assess the SCFA synthesis potential of HGM samples from several public metagenomic studies. We report baseline and variance for Community Phenotype Indices calculated for SCFAs production capabilities in 16S metagenomic samples of intestinal microbiota from two large national cohorts (American Gut Project, UK twins), the Hadza hunter-gatherers, and the young children cohort of infants with high-risk for type 1 diabetes. We further linked the predicted SCFA metabolic capabilities with available SCFA concentrations both for in vivo fecal samples and in vitro fermentation samples from previous studies. Finally, we analyzed differential representation of individual SCFA pathway genes across several WGS metagenomic datasets. The obtained collection of SCFA pathway genes and phenotypes enables the predictive metabolic phenotype profiling of HGM datasets and enhances the in silico methodology to study cross-feeding interactions in the gut microbiomes.

Metabolic engineering of threonine catabolism enables Saccharomyces cerevisiae to produce propionate under aerobic conditions.

Propionate is widely used as a preservative in the food and animal feed industries. Propionate is currently produced by petrochemical processes, and fermentative production of propionate remains challenging. In this study, a synthetic propionate pathway was constructed in the budding yeast Saccharomyces cerevisiae, for propionate production under aerobic conditions. Through expression of tdcB and aldH from Escherichia coli and kivD from Lactococcus lactis, L-threonine was converted to propionate via 2-ketobutyrate and propionaldehyde. The resulting yeast aerobically produced 0.21 g L-1 propionate from glucose in a shake flask. Subsequent overexpression of pathway genes and elimination of competing pathways increased propionate production to 0.37 g L-1 . To further increase propionate production, carbon flux was pulled into the propionate pathway by weakened expression of pyruvate kinase (PYK1), together with overexpression of phosphoenolpyruvate carboxylase (ppc). The final propionate production reached 1.05 g L-1 during fed-batch fermentation in a fermenter. In this work, a yeast cell factory was constructed using synthetic biology and metabolic engineering strategies to enable propionate production under aerobic conditions. Our study demonstrates engineered S. cerevisiae as a promising alternative for the production of propionate and its derivatives.

The screening of early warning indicators and microbial community of chicken manure thermophilic digestion at high organic loading rate

Anaerobic digestion of chicken manure (CM) with high nitrogen content is easily suffered inhibition of ammonia, especially under thermophilic and high organic loading rate (OLR) condition. To screen the early warning indicators for instability, thermophilic digestion of CM was conducted in a bench-scale reactor (working volume 55 L) under OLR from 1.0 to 6.0 g VS/(L·d). Microbial community analysis was used to reveal the mechanism of instability at high OLR. The system collapsed at an OLR of 6.0 g VS/(L·d). The initial ammonia inhibition evoked serious acidification, subsequently, the dual suppression of free volatile fatty acids and free ammonia caused the final collapse. The most likely mechanism of iso-butyrate degradation was propionate pathway. The ratio of n-butyrate to iso-butyrate was screened as an early warning indicator with the threshold of above 0.45. The high OLR of 6.0 g VS/(L·d) broke the balance of syntrophic oxidation bacteria (norank_o__MBA03, Gelria, and norank_o__D8A-2), hydrogenotrophic methanogens (Methanothermobacter) and fermentative and acid-producing bacteria (Ruminiclostridium_1, Bacteroides, Defluviitalea, norank_o__Clostridia, and norank_o__M55-D21). The non-functional acid tolerant bacteria (Defluviitoga, norank_f__Family_XI, Corynebacterrium_1, Caldicoprobacter, Lactobacillus, and Erysipelothrix) became the dominant after the system collapsed.

Six Patients with MethylmalonicAcidemia and Their Outcome: A Literature Review and Case Series

Background: Methylmalonic acidemia (MMA) is a congenital disorder due to the defects in the propionate pathway. It results from a deficiency in methylmalonyl coenzyme A mutase or one of the steps of the synthesis of the cobalamin (B12) cofactors for the enzyme. There is deficiency of methylmalonylcoAmutase (MCM) in the classic MMA. It presents with severe metabolic acidosis in the first month of life, progressive failure to thrive, feeding problems, recurrent vomiting, dehydration, hepatomegaly, lethargy, seizures, and developmental delay. Quantitative analysis of urinary organic acid patterns by GC-MS is used in MMA diagnosis. Treatment with large doses of hydroxocobalamin is helpful in some cases of MMA. Case presentation:We Reported 6 patients with MMA with a variety of clinical manifestations and outcomes. Conclusion: The overall prognosis of classic MMA remains doubtful, whereas vitamin B12 responsive MMA has a reasonable outcome.

Tricarboxylic acid cycle enzyme activities in a mouse model of methylmalonic aciduria

Methylmalonic acidemia (MMA) is a propionate pathway disorder caused by dysfunction of the mitochondrial enzyme methylmalonyl-CoA mutase (MMUT). MMUT catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA, an anaplerotic reaction which feeds into the tricarboxylic acid (TCA) cycle. As part of the pathological mechanisms of MMA, previous studies have suggested there is decreased TCA activity due to a “toxic inhibition” of TCA cycle enzymes by MMA related metabolites, in addition to reduced anaplerosis. Here, we have utilized mitochondria isolated from livers of a mouse model of MMA (Mut-ko/ki) and their littermate controls (Ki/wt) to examine the amounts and enzyme functions of most of the TCA cycle enzymes. We have performed mRNA quantification, protein semi-quantitation, and enzyme activity quantification for TCA cycle enzymes in these samples. Expression profiling showed increased mRNA levels of fumarate hydratase in the Mut-ko/ki samples, which by contrast had reduced protein levels as detected by immunoblot, while all other mRNA levels were unaltered. Immunoblotting also revealed decreased protein levels of 2-oxoglutarate dehydrogenase and malate dehydrogenase 2. Interesting, the decreased protein amount of 2-oxoglutarate dehydrogenase was reflected in decreased activity for this enzyme while there is a trend towards decreased activity of fumarate hydratase and malate dehydrogenase 2. Citrate synthase, isocitrate dehydrogenase 2/3, succinyl-CoA synthase, and succinate dehydrogenase are not statistically different in terms of quantity of enzyme or activity. Finally, we found decreased activity when examining the function of methylmalonyl-CoA mutase in series with succinate synthase and succinate dehydrogenase in the Mut-ko/ki mice compared to their littermate controls, as expected. This study demonstrates decreased activity of certain TCA cycle enzymes and by corollary decreased TCA cycle function, but it supports decreased protein quantity rather than “toxic inhibition” as the underlying mechanism of action. SummaryMethylmalonic acidemia (MMA) is an inborn metabolic disorder of propionate catabolism. In this disorder, toxic metabolites are considered to be the major pathogenic mechanism for acute and long-term complications. However, despite optimized therapies aimed at reducing metabolite levels, patients continue to suffer from late complications, including metabolic stroke and renal insufficiency. Since the propionate pathway feeds into the tricarboxylic acid (TCA) cycle, we investigated TCA cycle function in a constitutive MMA mouse model. We demonstrated decreased amounts of the TCA enzymes, Mdh2 and Ogdh as semi-quantified by immunoblot. Enzymatic activity of Ogdh is also decreased in the MMA mouse model compared to controls. Thus, when the enzyme amounts are decreased, we see the enzymatic activity also decreased to a similar extent for Ogdh. Further studies to elucidate the structural and/or functional links between the TCA cycle and propionate pathways might lead to new treatment approaches for MMA patients.

THE INFLUENCE OF PROPIONATE ON THE SPECTRUM OF LONG-CHAIN FATTY ACIDS IN CERVICAL CELLS DURING DIS- AND NEOPLASTIC TRANSFORMATION

Background.Metabolic reprogramming of tumor cells is one of the leading links in carcinogenesis. The objective to confirm the hypothesis of a propionate pathway for the synthesis of long-chain acids with an odd number of carbon atoms in cervical cancer.Materials and methods.As samples for the study were biopsies of the colli uteri, from which a suspension of tumor cells was obtained (20 – cervical cancer, 20 – cervical intraepithelial neoplasia III grade, 18 – endocervical scraping from healthy women). The spectrum of fatty acids (FA) was analyzed before, after 24 hours incubation without and with 50 μmol/l propionic acid by the gas chromatography method.Results.Metabolism of FA is multidirectional in different locus in cervical cancer. In the locus of cervical cancer, most likely, there is a propionic pathway for the synthesis of FA using palmitate. In cervical intraepithelial neoplasia III grade, the metabolism of FA is another. Palmitic acid was used for synthesis stearate, which was metabolized to oleic acid.Сonclusion.The specificity of biochemical changes within one organ and one pathology has been revealed, which reflects the stage-by-stage development of the oncological process.

Current concepts in organic acidurias: understanding intra‐ and extracerebral disease manifestation

This review focuses on the pathophysiology of organic acidurias (OADs), in particular, OADs caused by deficient amino acid metabolism. OADs are termed classical if patients present with acute metabolic decompensation and multiorgan dysfunction or cerebral if patients predominantly present with neurological symptoms but without metabolic crises. In both groups, however, the brain is the major target. The high energy demand of the brain, the gate-keeping function of the blood-brain barrier, a high lipid content, vulnerable neuronal subpopulations, and glutamatergic neurotransmission all make the brain particularly vulnerable against mitochondrial dysfunction, oxidative stress, and excitotoxicity. In fact, toxic metabolites in OADs are thought to cause secondary impairment of energy metabolism; some of these toxic metabolites are trapped in the brain. In contrast to cerebral OADs, patients with classical OADs have an increased risk of multiorgan dysfunction. The lack of the anaplerotic propionate pathway, synergistic inhibition of energy metabolism by toxic metabolites, and multiple oxidative phosphorylation (OXPHOS) deficiency may best explain the involvement of organs with a high energy demand. Intriguingly, late-onset organ dysfunction may manifest even under metabolically stable conditions. This might be explained by chronic mitochondrial DNA depletion, increased production of reactive oxygen species, and altered gene expression due to histone modification. In conclusion, pathomechanisms underlying the acute disease manifestation in OADs, with a particular focus on the brain, are partially understood. More work is required to predict the risk and to elucidate the mechanism of late-onset organ dysfunction, extracerebral disease manifestation, and tumorigenesis.

Controlling the feed rate of glucose and propanol for the enhancement of erythromycin production and exploration of propanol metabolism fate by quantitative metabolic flux analysis

In this paper, several different fermentation experiments were designed to address whether modulating glucose and propanol feeds could benefit the production level of erythromycin during pilot plant (30 L) fermentation. Results showed that glucose feed rate (determined by a set high or low culture pH) had no effect on erythromycin production, indicating that glucose was not the limiting factor for erythromycin biosynthesis under these conditions. It was found that decreasing glucose feed could stimulate the consumption of propanol, and the high erythromycin production (12.49 ± 0.50 mg ml⁻¹) was achieved by controlling the feed rates of glucose and propanol. The quantitative metabolic flux analysis disclosed that high propanol consumption increased the pool size of propionyl-CoA (~2.147 mmol g⁻¹ day⁻¹) and methylmalonyl-CoA (~1.708 mmol g⁻¹ day⁻¹). It was also found that 45-77 % of the propanol went into the TCA cycle which strengthened the conclusion that blocking the propionate pathway to TCA cycle could lead to a significant increase in erythromycin production in carbohydrate-based media (Reeves et al. Ind Microbiol Biotechnol 7:600-609, 2006). In addition, the results also suggested that a relative low intracellular ATP level resulting from low glucose feed did not limit the erythromycin biosynthesis, and a relatively high NADPH should be beneficial for erythromycin biosynthesis.

Fermentation characteristics of Fusariumoxysporum grown on acetate

In this study, the growth characteristics of Fusariumoxysporum were evaluated in minimal medium using acetate or different mixtures of acetate and glucose as carbon source. The minimum inhibitory concentration (MIC) of acetic acid that F.oxysporum cells could tolerate was 0.8%w/v while glucose was consumed preferentially to acetate. The activity of isocitrate lyase was high when cells were grown on acetate and acetate plus glucose indicating an activation of the glyoxylate cycle. Investigation of the metabolic fingerprinting and footprinting revealed higher levels of intracellular and extracellular TCA cycle intermediates when F.oxysporum cells were grown on mixtures of acetate and glucose compared to growth on only glucose. Our data support the hypothesis that a higher flux through TCA cycle during acetate consumption could significantly increase the pool of NADH, resulting in the activation of succinate–propionate pathway which consumes reducing power (NADH) via conversion of succinate to propionyl-CoA and produce propionate.

Marenzelleria viridis (Verrill, 1873) (Polychaeta), a new benthic species within European coastal waters Some metabolic features

The polychaete Marenzelleria viridis (Verrill, 1873) is a recent invader of European brackish waters (North Sea and Baltic Sea). As a member of the coastal infaunal community, dwelling in permanent tubes, M. viridis is frequently subjected to extreme environmental conditions, such as periods with restricted oxygen availability. When exposed to experimental anoxia, M. viridis follows in general the metabolic pathways exploited by many marine invertebrates. However, some differences become obvious. In contrast to most polychaetes, M. viridis used phospho- l-arginine for short-term energy provision and octopine was found to be the only end-product of cytosolic glycolysis. Alanopine and strombine, which were also measured were only found in traces. This corresponds with the relatively high activity of the octopine dehydrogenase (18 U g −1 wet wt.), whereas the activities of other known pyruvate oxidoreductases were clearly lower or almost zero. Remarkable differences in the mode of anaerobic energy production were not found in populations with different salinity regimes from the Baltic Sea (Darss-Zingst bodden chain, S 8%.) and the North Sea (Ems estuary, S 15%.). Although glycine concentrations were clearly higher in the North Sea population, strombine did not occur as end-product of anaerobic glycolysis. However, alanine was accumulated to a higher amount in the North Sea population, albeit the concentrations of alanine were nearly the same in both populations. Compared to other marine polychaetes, M. viridis (North Sea and Baltic) showed only a moderate resistance against anoxic conditions. The reasons appear to be the relatively low glycogen content and insufficient reduction of the energy demand, indicated by the strong decrease in the energy charge after 24 h (0.82 to 0.70). Furthermore, M. viridis switched much earlier to energy production via the ‘propionate pathway’ and propionate was first detected in the incubation medium three hours after onset of anoxia. However, in M. viridis anaerobic energy production started at a very low oxygen tension (critical p O 2 at about 2 kPa) which indicates a very efficient use of oxygen even at severe hypoxia.

Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant.

This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution 13C NMR. The oxidation of [1,2,3-13C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS-) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The 13C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure (TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3-13C]propionate or [2-13C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.

Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy.

High-resolution 13C n.m.r. spectroscopy has been used to examine propionate metabolism in the perfused rat heart. A number of tricarboxylic acid (TCA) cycle intermediates are observable by 13C n.m.r. in hearts perfused with mixtures of pyruvate and propionate. When the enriched 13C-labelled nucleus originates with pyruvate, the resonances of the intermediates appear as multiplets due to formation of multiply-enriched 13C-labelled isotopomers, whereas when the 13C-labelled nucleus originates with propionate, these same intermediates appear as singlets in the 13C spectrum since entry of propionate into the TCA cycle occurs via succinyl-CoA. An analysis of the isotopomer populations in hearts perfused with [3-13C]pyruvate plus unlabelled propionate indicates that about 27% of the total pyruvate pool available to the heart is derived directly from unlabelled propionate. This was substantiated by perfusing a heart for 2 h with [3-13C]propionate as the only available exogenous substrate. Under these conditions, all of the propionate consumed by the heart, as measured by conventional chemical analysis, ultimately entered the oxidative pathway as [2-13C] or [3-13C]pyruvate. This is consistent with entry of propionate into the TCA cycle intermediate pools as succinyl-CoA and concomitant disposal of malate to pyruvate via the malic enzyme. 13C resonances arising from enriched methylmalonate and propionylcarnitine are also detected in hearts perfused with [3-13C] or [1-13C]propionate which suggests that 13C n.m.r. may be useful as a non-invasive probe in vivo of metabolic abnormalities involving the propionate pathway, such as methylmalonic aciduria or propionic acidaemia.

Propionate metabolism by cultured skin fibroblasts from normal individuals and patients with methylmalonicaciduria and propionicacidemia

The incorporation of [1- 14C]propionate into trichloroacetic acid-precipitable material was found to be greater in normal fibroblasts than in cells cultured from patients with PrAE and MMAU. In MMAU cells, the metabolic pathway leading into the TCA cycle appeared to be completely blocked. In PrAE cells, however, trace amounts of labeled TCA cycle intermediates and lactic acid were detected, indicating an incomplete block in the propionate pathway. A large accumulation of labeled long-chain fatty acids by affected cell lines points to a possible role of fatty acids in pathogenesis of these disorders.

Inborn errors of cobalamin metabolism: effect of cobalamin supplementation in culture on methylmalonyl CoA mutase activity in normal and mutant human fibroblasts.

We have examined the effect of addition of hydroxocobalamin to growth medium on the activity of the adenosylcobalamin-requiring enzyme methylmalonyl CoA mutase in normal human fibroblasts and in mutant human fibroblasts derived from patients with inherited methylmalonicacidemia. The mutant cell lines were assigned to four distinct genetic complementation groups (cbl A, cbl B, cbl C, and cbl D), each deficient in some step in the synthesis of adenosylcobalamin from hydroxocobalamin. After control cells were grown in cobalamin-supplemented medium, mutase holoenzyme activitiy increased markedly in a time- and concentration-dependent fashion. Growth in cobalamin-supplemented medium had no effect on mutase activity in some mutant lines belonging to the cbl B group, while activity increased severalfold in other cbl B mutants and in all cbl A, cbl C, and cbl D mutants examined, although mutase activity was still less than 10% of control. Comparison of mutase holoenzyme activity and total propionate pathway activity suggests that enhancement of mutase activity in mutant cells after cobalamin supplementation to values 5--10% of control may be sufficient to overcome the inherited metabolic block and to restore total pathway activity to normal.

Rapid prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism: a sensitive assay using cultured cells.

A sensitive, reliable, and easily performed procedure is described for the prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism using cultured amniotic cells and skin fibroblasts. With this assay, control fibroblast lines incorporated a mean of 6.89 nanoatoms 14C/mg protein from [1-14C]propionate into trichloroacetic acid (TCA)-precipitable cell material in 10 h. Twenty-five mutant fibroblast lines from patients with propionicacidemia or one of the methylmalonicacidemias fixed 0.04 to 0.93 nanoatoms 14C/mg. Considerable variation was observed, both among and within discrete mutant classes, with respect to the residual amount of propionate pathway activity, possibly reflecting further molecular heterogeneity in these disorders. We applied this procedure to cultured amniotic cells from controls and 4 midtrimester pregnancies at risk for methylmalonicacidemia and diagnosed one fetus with a methylmalonyl CoA apomutase defect and 3 fetuses which were unaffected.

Carbon dioxide fixation in marine invertebrates—V. Rate and pathway in the oyster

1. 1. Isolated oyster mantle tissue was incubated at 25° with NaH 14CO 3 of high specific activity to provide a medium closely resembling natural sea water in salinity and total carbon dioxide concentration. 2. 2. The rate of CO 2 fixation into six acids of the citric acid cycle was determined as 0·088 μmole/g fresh tissue/hr in the first few seconds; in periods of 11·4 sec to 60 min, the apparent rate was a constant 0·016 μmole/g/hr; the higher rate was 1·3 per cent of the rate of oxygen consumption by this tissue. 3. 3. The pattern of labeling of the acids indicated that malic acid was the initial major product of CO 2 fixation; spectrophotometric evidence for the presence of a malic enzyme in the supernatant of centrifuged homogenates was found. 4. 4. Succinic acid accumulated radioactivity at the highest rate, which suggested additional influx of CO 2 via the propionate pathway or the presence of an active fumarate reductase, or both.