mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria.

The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [18F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.

New Perspectives in Dried Blood Spot Biomarkers for Lysosomal Storage Diseases.

Dried blood spots (DBSs) biomarkers are convenient for monitoring for specific lysosomal storage diseases (LSDs), but they could have relevance for other LSDs. To determine the specificity and utility of glycosphingolipidoses biomarkers against other LSDs, we applied a multiplexed lipid liquid chromatography tandem mass spectrometry assay to a DBS cohort of healthy controls (n = 10) and Gaucher (n = 4), Fabry (n = 10), Pompe (n = 2), mucopolysaccharidosis types I-VI (n = 52), and Niemann-Pick disease type C (NPC) (n = 5) patients. We observed no complete disease specificity for any of the markers tested. However, comparison among the different LSDs highlighted new applications and perspectives of the existing biomarkers. We observed elevations in glucosylceramide isoforms in the NPC and Gaucher patients relative to the controls. In NPC, there was a greater proportion of C24 isoforms, giving a specificity of 96-97% for NPC, higher than 92% for the NPC biomarker N-palmitoyl-O-phosphocholineserine ratio to lyso-sphingomyelin. We also observed significantly elevated levels of lyso-dihexosylceramide in Gaucher and Fabry disease as well as elevated lyso-globotriaosylceramide (Lyso-Gb3) in Gaucher disease and the neuronopathic forms of Mucopolysaccharidoses. In conclusion, DBS glucosylceramide isoform profiling has increased the specificity for the detection of NPC, thereby improving diagnostic accuracy. Low levels of lyso-lipids can be observed in other LSDs, which may have implications in their disease pathogenesis.

Human ultrarare genetic disorders of sulfur metabolism demonstrate redundancies in H2S homeostasis.

Regulation of H2S homeostasis in humans is poorly understood. Therefore, we assessed the importance of individual enzymes in synthesis and catabolism of H2S by studying patients with respective genetic defects. We analyzed sulfur compounds (including bioavailable sulfide) in 37 untreated or insufficiently treated patients with seven ultrarare enzyme deficiencies and compared them to 63 controls. Surprisingly, we observed that patients with severe deficiency in cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) - the enzymes primarily responsible for H2S synthesis - exhibited increased and normal levels of bioavailable sulfide, respectively. However, an approximately 21-fold increase of urinary homolanthionine in CBS deficiency strongly suggests that lacking CBS activity is compensated for by an increase in CSE-dependent H2S synthesis from accumulating homocysteine, which suggests a control of H2S homeostasis in vivo. In deficiency of sulfide:quinone oxidoreductase - the first enzyme in mitochondrial H2S oxidation - we found normal H2S concentrations in a symptomatic patient and his asymptomatic sibling, and elevated levels in an asymptomatic sibling, challenging the requirement for this enzyme in catabolizing H2S under physiological conditions. Patients with ethylmalonic encephalopathy and sulfite oxidase/molybdenum cofactor deficiencies exhibited massive accumulation of thiosulfate and sulfite with formation of large amounts of S-sulfocysteine and S-sulfohomocysteine, increased renal losses of sulfur compounds and concomitant strong reduction in plasma total cysteine. Our results demonstrate the value of a comprehensive assessment of sulfur compounds in severe disorders of homocysteine/cysteine metabolism and provide evidence for redundancy and compensatory mechanisms in the maintenance of H2S homeostasis.

Rapid, proteomic urine assay for monitoring progressive organ disease in Fabry disease.

BACKGROUND: Fabry disease is a progressive multisystemic disease, which affects the kidney and cardiovascular systems. Various treatments exist but decisions on how and when to treat are contentious. The current marker for monitoring treatment is plasma globotriaosylsphingosine (lyso-Gb3), but it is not informative about the underlying and developing disease pathology. METHODS: We have created a urine proteomic assay containing a panel of biomarkers designed to measure disease-related pathology which include the inflammatory system, lysosome, heart, kidney, endothelium and cardiovascular system. Using a targeted proteomic-based approach, a series of 40 proteins for organ systems affected in Fabry disease were multiplexed into a single 10 min multiple reaction monitoring Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) assay and using only 1 mL of urine. RESULTS: Six urinary proteins were elevated in the early-stage/asymptomatic Fabry group compared with controls including albumin, uromodulin, α1-antitrypsin, glycogen phosphorylase brain form, endothelial protein receptor C and intracellular adhesion molecule 1. Albumin demonstrated an increase in urine and could indicate presymptomatic disease. The only protein elevated in the early-stage/asymptomatic patients that continued to increase with progressive multiorgan involvement was glycogen phosphorylase brain form. Podocalyxin, fibroblast growth factor 23, cubulin and Alpha-1-Microglobulin/Bikunin Precursor (AMBP) were elevated only in disease groups involving kidney disease. Nephrin, a podocyte-specific protein, was elevated in all symptomatic groups. Prosaposin was increased in all symptomatic groups and showed greater specificity (p<0.025-0.0002) according to disease severity. CONCLUSION: This work indicates that protein biomarkers could be helpful and used in conjunction with plasma lyso-Gb3 for monitoring of therapy or disease progression in patients with Fabry disease.

PDXK mutations cause polyneuropathy responsive to pyridoxal 5'-phosphate supplementation.

OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. RESULTS: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240.

Disorders affecting vitamin B6 metabolism.

Vitamin B6 is present in our diet in many forms, however, only pyridoxal 5'-phosphate (PLP) can function as a cofactor for enzymes. The intestine absorbs nonphosphorylated B6 vitamers, which are converted by specific enzymes to the active PLP form. The role of PLP is enabled by its reactive aldehyde group. Pathways reliant on PLP include amino acid and neurotransmitter metabolism, folate and 1-carbon metabolism, protein and polyamine synthesis, carbohydrate and lipid metabolism, mitochondrial function and erythropoiesis. Besides the role of PLP as a cofactor B6 vitamers also play other cellular roles, for example, as antioxidants, modifying expression and action of steroid hormone receptors, affecting immune function, as chaperones and as an antagonist of Adenosine-5'-triphosphate (ATP) at P2 purinoceptors. Because of the vital role of PLP in neurotransmitter metabolism, particularly synthesis of the inhibitory transmitter γ-aminobutyric acid, it is not surprising that various inborn errors leading to PLP deficiency manifest as B6 -responsive epilepsy, usually of early onset. This includes pyridox(am)ine phosphate oxidase deficiency (a disorder affecting PLP synthesis and recycling), disorders affecting PLP import into the brain (hypophosphatasia and glycosylphosphatidylinositol anchor synthesis defects), a disorder of an intracellular PLP-binding protein (PLPBP, previously named PROSC) and disorders where metabolites accumulate that inactivate PLP, for example, ALDH7A1 deficiency and hyperprolinaemia type II. Patients with these disorders can show rapid control of seizures in response to either pyridoxine and/or PLP with a lifelong dependency on supraphysiological vitamin B6 supply. The clinical and biochemical features of disorders leading to B6 -responsive seizures and the treatment of these disorders are described in this review.

Enhanced pre-operative axillary staging using intradermal microbubbles and contrast-enhanced ultrasound to detect and biopsy sentinel lymph nodes in breast cancer: a potential replacement for axillary surgery.

OBJECTIVE: To compare the experience of four UK Centres in the use of intradermal microbubbles and contrast enhanced ultrasound (CEUS) to pre-operatively identify and biopsy sentinel lymph nodes (SLN) in patients with breast cancer. METHODS: In all centres, breast cancer patients had a microbubble/CEUS SLN core biopsy prior to axillary surgery and patients in Centres 1 and 2 had a normal greyscale axillary ultrasound. Data were collected between 2010 and 2016; 1361 from Centre 1 (prospective, sequential), 376 from Centre 2 (retrospective, sequential), 121 from Centre 3 (retrospective, selected) and 48 from Centre 4 (prospective, selected). RESULTS: SLN were successfully core biopsied in 80% (Centre 1), 79.6% (Centre 2), 77.5% (Centre 3) and 88% (Centre 4). The sensitivities to identify all SLN metastases were 46.9% [95% confidence intervals (CI) (39.4-55.1)], 52.5% [95% CI (39.1-65.7)], 46.4% [95% CI (27.5-66.1)] and 45.5% [95% CI (16.7-76.6)], respectively. The specificities were 99.7% [95% CI (I98.9-100)], 98.1% [95% CI (94.5-99.6)], 100% [95% CI (93.2-100%)] and 96.3% [95% CI (81-99.9)], respectively.The negative predictive values were 87.0% [95% CI (84.3-89.3)], 84.5% [95% CI (78.4-89.5)], 86.9% [95% CI (82.4-90.3)] and 86.2% [95% CI (78.4-91.5)], respectively. At Centres 1 and 2, 12/730 (1.6%) and 7/181 (4%), respectively, of patients with a benign microbubble/CEUS SLN core biopsy had two or more lymph node (LN) macrometastases found at the end of primary surgical treatment. CONCLUSION: The identification and biopsy of SLN using CEUS is a reproducible technique. Advances in knowledge: In the era of axillary conservation, microbubble/CEUS SLN core biopsy has the potential to succeed surgical staging of the axilla.

Micronutrients: Speculation on Inborn Errors, Nutrigenomics, Evolution, the Microbiome, and Nutritional Immunity

Abstract Many micronutrients or cofactors derived from micronutrients are highly reactive, hence their role in catalysis of reactions by enzymes. The concentration of cofactors has to be kept low to avoid unwanted reactions while allowing them to bind to the (apo)enzymes that need them. A new disorder causing B6-responsive epilepsy (proline synthetase cotranscribed bacterial homologue deficiency) is probably due to the absence of an important intracellular pyridoxal phosphate chaperone. The availability of some micronutrients varies by orders of magnitude in different geographical areas. Selenium is both essential and toxic, and during evolution, different populations have had to adapt to this differing availability. An "inborn error of metabolism (IEM)" in a low selenium area of China may be a selective advantage in a high selenium area and vice versa; the concept of nutrigenomics is an important one for micronutrients. The gut flora may make an important contribution to vitamin synthesis. This is difficult to study, but experiments can be undertaken with the nematode, Caenorhabditis elegans (with or without an IEM) and a single clone of Escherichia coli (with or without an IEM) as food and gut flora. This model shows that the gut microbiome can have profound influences on the folate cycle and associated vitamins. Our innate immune system makes use of the micronutrient requirements of pathogens and can deprive a pathogen of essential micronutrient(s) or expose it to toxic levels. It is not surprising, therefore, that some mutations affecting the way the host handles micronutrients can confer an advantage in resistance to infection and this may have acted as a selective advantage during evolution. This will be discussed by reference to the relationship of inborn errors to resistance to malaria. Conversely, other inborn errors of micronutrient metabolism are likely to make it more difficult for the host to use nutritional immunity to fight infection; this probably accounts for the list of infections that are more serious in patients with hereditary haemochromatosis.

Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans.

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.

Predictive factors for invasive cancer in surgical specimens following an initial diagnosis of ductal carcinoma in situ after stereotactic vacuum-assisted breast biopsy in microcalcification-only lesions.

PURPOSE: The aim of this study was to determine the incidence of invasive breast carcinoma in patients with preoperative diagnosis of ductal carcinoma in situ (DCIS) by stereotactic vacuum-assisted biopsy (SVAB) performed for microcalcification-only lesions, and to identify the predictive factors of invasion. METHODS: From 2000 to 2010, the records of 353 DCIS patients presenting with microcalcification-only lesions who underwent SVAB were retrospectively reviewed. The mammographic size of microcalcification cluster, presence of microinvasion within the cores, the total number of calcium specks, and the number of calcium specks within the retrieved core biopsy specimen were recorded. Patients were grouped as those with or without invasion in the final pathologic report, and variables were compared between the two groups. RESULTS: The median age was 58 years (range, 34-88 years). At histopathologic examination of the surgical specimen, 63 of 353 patients (17.8%) were found to have an invasive component, although SVAB cores had only shown DCIS preoperatively. The rate of underestimation was significantly higher in patients with microcalcification covering an area of 40 mm or more, in the presence of microinvasion at biopsy, and in cases where less than 40% of the calcium specks were removed from the lesion. CONCLUSION: Invasion might be underestimated in DCIS cases diagnosed with SVAB performed for microcalcification-only lesions, especially when the mammographic size of calcification is equal to or more than 40 mm or if microinvasion is found within the biopsy specimen and less than 40% of the calcifications are removed. At least 40% of microcalcification specks should be removed from the lesion to decrease the rate of underestimation with SVAB.

Liver disease in infancy caused by oxysterol 7 α-hydroxylase deficiency: successful treatment with chenodeoxycholic acid.

A child of consanguineous parents of Pakistani origin developed jaundice at 5 weeks and then, at 3 months, irritability, a prolonged prothrombin time, a low albumin, and episodes of hypoglycaemia. Investigation showed an elevated alanine aminotransferase with a normal γ-glutamyl-transpeptidase. Analysis of urine by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) showed that the major peaks were m/z 480 (taurine-conjugated 3ß-hydroxy-5-cholenoic acid) and m/z 453 (sulphated 3ß-hydroxy-5-cholenoic acid). Analysis of plasma by gas chromatography-mass spectrometry (GC-MS) showed increased concentrations of 3ß-hydroxy-5-cholenoic acid, 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol, indicating oxysterol 7 α-hydroxylase deficiency. The patient was homozygous for a mutation (c.1249C>T) in CYP7B1 that alters a highly conserved residue in oxysterol 7 α-hydroxylase (p.R417C) - previously reported in a family with hereditary spastic paraplegia type 5. On treatment with ursodeoxycholic acid (UDCA), his condition was worsening, but on chenodeoxycholic acid (CDCA), 15 mg/kg/d, he improved rapidly. A biopsy (after 2 weeks on CDCA), showed a giant cell hepatitis, an evolving micronodular cirrhosis, and steatosis. The improvement in liver function on CDCA was associated with a drop in the plasma concentrations and urinary excretions of the 3ß-hydroxy-Δ5 bile acids which are considered hepatotoxic. At age 5 years (on CDCA, 6 mg/kg/d), he was thriving with normal liver function. Neurological development was normal apart from a tendency to trip. Examination revealed pes cavus but no upper motor neuron signs. The findings in this case suggest that CDCA can reduce the activity of cholesterol 27-hydroxylase - the first step in the acidic pathway for bile acid synthesis.

A new method for the rapid diagnosis of protein N-linked congenital disorders of glycosylation.

The Congenital Disorders of Glycosylation (CDG) are a devastating group of genetic disorders that encompass a spectrum of glycosylation defects and are characterized by the underglycosylation of or the presence of abnormal glycans on glycoproteins. The N-linked CDG disorders (Type I and II) are usually diagnosed in chemical pathology laboratories by an abnormal serum transferrin isoelectric focusing (IEF) pattern. Transferrin has been the protein of choice for CDG analysis because it is well characterized, highly abundant, and easily detected in plasma. However, IEF provides limited information on the glycosylation defect and requires a separate and extensive glycan analysis to diagnose CDG Type II. We have therefore developed a simple bead-based immunoaffinity and mass spectrometry-based assay to address these issues. Our method uses immuno-purified transferrin and proteolytic digestion followed by a rapid 30 min mass spectral analysis and allows us to identify both micro- and macroheterogeneity of transferrin by sequencing of peptides and glycopeptides. In summary, we have developed a simple, rapid test for N-linked glycosylation disorders that is a significant improvement on existing laboratory tests currently used for investigating defective N-linked glycosylation.

Factors that impact the upgrading of atypical ductal hyperplasia.

PURPOSE: The purpose of this study was to identify the factors that may have an impact on upgrading atypical ductal hyperplasia (ADH) lesions to malignancy. MATERIALS AND METHODS: Between February 1999 and December 2010, the records of 150 ADH lesions that had been biopsied were retrospectively reviewed. The biopsy types included 11-gauge stereotactic vacuum-assisted biopsy (SVAB) (n=102) and ultrasonography (US)-guided 14-gauge automated biopsy (n=48). The patients were divided into two groups: those who had cancer in the final pathology and those who did not. Variables associated with underestimation of ADH lesions were compared between the groups. RESULTS: The underestimation rates according to the biopsy types were 41.7% (20/48) for the US-guided 14-gauge automated biopsy and 20.6% (21/102) for the 11-gauge SVAB (P = 0.007). The rate of underestimation was significantly higher in lesions greater than 7 mm than it was in smaller lesions, with both US-guided 14-gauge automated biopsy and 11-gauge SVAB (P = 0.024 and P = 0.042, respectively). The rate of underestimation was significantly higher with the 11-gauge SVAB (P = 0.025) in lesions that were suspicious (R4) and highly suggestive of malignancy (R5) than in those that were probably benign (R3). CONCLUSION: The underestimation rate in ADH lesions was significantly higher with US-guided 14-gauge automated biopsy compared to the 11-gauge SVAB. The underestimation rate was also significantly higher in lesions greater than 7 mm regardless of the biopsy type, and in lesions biopsied using SVAB that were regarded as suspicious (R4) or highly suggestive of malignancy (R5) on imaging.

Preoperative needle biopsy of sentinel lymph nodes using intradermal microbubbles and contrast-enhanced ultrasound in patients with breast cancer.

OBJECTIVE: The purpose of this study was to assess whether sentinel lymph nodes (SLNs) that undergo targeted needle biopsy after identification by contrast-enhanced ultrasound (CEUS) using intradermally injected microbubbles results in more node-positive breast cancer patients being diagnosed preoperatively. Furthermore, we sought to determine whether the addition of CEUS to gray-scale sonography of the axilla reduces the number of patients having axillary lymph node (ALN) dissection as a second procedure. SUBJECTS AND METHODS: Intradermal microbubble injection was performed in 136 breast cancer patients who had no abnormal ALNs on routine gray-scale axillary sonography. When an enhancing ALN was visualized, percutaneous sonography-guided fine-needle aspiration cytology or core needle biopsy was performed. Depending on the biopsy results, patients underwent SLN biopsy or ALN dissection. If the putative SLN biopsy was positive or a biopsy tract was seen in the excised SLN, the procedure was defined as successful. RESULTS: SLNs were identified and biopsied in 126 of the 136 cases (93%). Seventeen patients had positive sonography-guided biopsy results (13%) and were treated with immediate ALN dissection. In seven patients, the biopsied node was the only positive node. The remaining 109 patients underwent SLN biopsy. In nine cases (8%), a positive lymph node was identified. Four of these false-negative cases had only micrometastases. CONCLUSION: SLNs can be identified and biopsied using CEUS to increase the accuracy of preoperative axillary staging. If the needle biopsy result is negative, conventional SLN biopsy is indicated.

Causes of failure in removing calcium in microcalcification-only lesions using 11-gauge stereotactic vacuum-assisted breast biopsy.

PURPOSE: The aim of this study was to determine the causes and rate of failure in removing calcification in microcalcification-only lesions using 11-gauge stereotactic vacuum-assisted breast biopsy. MATERIALS AND METHODS: In total, 1365 microcalcification-only lesions were included in this study. The breast biopsy database was reviewed retrospectively. The biopsies were divided into two groups based on whether the specimen X-ray showed calcium within the cores. Breast composition, lesion size, calcification distribution, density on mammography, and the number of specimens were compared between the two groups. RESULTS: In 11 (0.8%) biopsies, no calcium in the specimen radiography could be identified. Re-biopsy was performed in five cases. The initial biopsy result was unchanged at the second biopsy in three cases containing calcium, while in the other two cases, a benign biopsy result was upgraded to atypical ductal hyperplasia and ductal carcinoma in situ, respectively. In six cases, the biopsy was not repeated despite the absence of calcium in the specimen X-ray. In three of these cases, calcifications were reported histopathologically and deemed to be too small to be identified on specimen X-ray. In two of six patients, sufficient information was found in the cores without microcalcification to indicate the need for surgery. One patient refused re-biopsy. A statistically significant higher failure rate was observed in low-density calcification compared with intermediate or high-density calcification on mammography. CONCLUSION: The failure to retrieve microcalcification is uncommon when an 11-gauge vacuum-assisted breast biopsy is used. Low-density calcifications have a higher rate of failure. In cases in which no calcium is observed in specimen radiography, repeated biopsy is recommended.

Urinary AASA excretion is elevated in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency.

Analysis of α-aminoadipic semialdehyde is an important tool in the diagnosis of antiquitin deficiency (pyridoxine-dependent epilepsy). However continuing use of this test has revealed that elevated urinary excretion of α-aminoadipic semialdehyde is not only found in patients with pyridoxine-dependent epilepsy but is also seen in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency. This should be taken into account when interpreting the laboratory data. Sulphite was shown to inhibit α-aminoadipic semialdehyde dehydrogenase in vitro.

Percutaneous removal of sentinel lymph nodes in a swine model using a breast lesion excision system and contrast-enhanced ultrasound.

OBJECTIVES: To investigate the feasibility of percutaneous removal of the entire sentinel lymph node (SLN) in an animal model using a breast lesion excision system after identifying these nodes using contrast-enhanced ultrasound (CEUS) and intradermal microbubbles. METHODS: Animal studies approval was obtained. SLNs were identified using CEUS and intradermal injection of microbubbles in two young pigs. Microbubbles were mixed with blue dye and injected around the mammary papillae to access lymphatic drainage to the superficial inguinal lymph nodes. When enhancing nodes were identified, the breast lesion excision system (BLES) was used to remove these nodes percutaneously. Both animals then underwent surgical lymph node dissection. Histopathological examination of all the samples was performed. RESULTS: Removal of the entire SLN was successful in three groins in the pigs. All three nodes were stained with blue dye. No other stained nodes were observed in the node dissection specimens. The nodal architecture of removed lymph nodes was well preserved on microscopy. There were no signs of excess trauma within the biopsy bed. CONCLUSION: The results obtained from the swine model demonstrated that it is feasible to remove the entire SLN percutaneously under the guidance of CEUS and microbubbles. KEY POINTS: Intradermal injection of microbubbles and CEUS can identify sentinel lymph nodes • Ultrasound could then guide percutaneous removal of intact and complete SLNs • We have shown this was feasible in pigs but not yet in humans • This technique may eventually have the potential to reduce futile SLN biopsies.

Bile acid-CoA ligase deficiency--a new inborn error of bile acid metabolism.

Born at 27 weeks gestation, a child of consanguineous parents of Pakistani origin required prolonged parenteral nutrition. She developed jaundice, with extensive fibrosis and architectural distortion at liver biopsy; jaundice resolved with supportive care. Serum γ-glutamyl transpeptidase values were within normal ranges. The bile acids in her plasma and urine were >85% unconjugated (non-amidated). Two genes encoding bile-acid amidation enzymes were sequenced. No mutations were found in BAAT, encoding bile acid-CoA : aminoacid N-acyl transferase. The patient was homozygous for the missense mutation c.1012C > T in SLC27A5, predicted to alter a highly conserved amino-acid residue (p.H338Y) in bile acid-CoA ligase (BACL). She also was homozygous for the missense mutation c.1772A > G in ABCB11, predicted to alter a highly conserved amino-acid residue (p.N591S) in bile salt export pump (BSEP). BACL is essential for reconjugation of bile acids deconjugated by gut bacteria, and BSEP is essential for hepatocyte-canaliculus export of conjugated bile acids. A female sibling born at term had the same bile-acid phenotype and SLC27A5 genotype, without clinical liver disease. She was heterozygous for the c.1772A > G ABCB11 mutation. This is the first report of a mutation in SLC27A5. The amidation defect may have contributed to cholestatic liver disease in the setting of prematurity, parenteral nutrition, and homozygosity for an ABCB11 mutation.

Pyridoxal 5'-phosphate in cerebrospinal fluid; factors affecting concentration.

Analysis of pyridoxal 5'-phosphate (PLP) concentration in 256 cerebrospinal fluid (CSF) samples from patients with neurological symptoms showed that the variance is greater than indicated by previous studies. The age-related lower reference limit has been revised to detect inborn errors of metabolism that lead to PLP depletion without a high false positive rate: < 30 days, 26 nmol/L; 30 days to 12 months, 14 nmol/L; 1-2 years, 11 nmol/L; > 3 years, 10 nmol/L. Inborn errors leading to PLP concentrations below these values include pyridoxine-dependent epilepsy due to antiquitin deficiency, and molybdenum cofactor deficiency that leads to the accumulation of sulfite, a nucleophile capable of reacting with PLP. Low PLP levels were also seen in a group of children with transiently elevated urinary excretion of sulfite and/or sulfocysteine, suggesting that there may be other situations in which sulfite accumulates and inactivates PLP. There was no evidence that seizures or the anticonvulsant drugs prescribed for patients in this study led to significant lowering of CSF PLP. A small proportion of patients receiving L-dopa therapy were found to have a CSF PLP concentration below the appropriate reference range. This may have implications for monitoring and treatment. A positive correlation was seen between the CSF PLP and 5-methyl-tetrahydrofolate (5-MTHF) and tetrahydrobiopterin (BH(4)) concentrations. All are susceptible to attack by nucleophiles and oxygen-derived free-radicals, and CSF has relatively low concentrations of other molecules that can react with these compounds. Further studies of CSF PLP levels in a wide range of neurological diseases might lead to improved understanding of pathogenesis and possibilities for treatment.

Preoperative sentinel node identification with ultrasound using microbubbles in patients with breast cancer.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is the standard procedure for axillary staging in early breast cancer. Lymphatic imaging after peritumoral microbubble injection has been described in animal models. The aim of this study was to identify and localize SLNs preoperatively by contrast-enhanced sonography after intradermal injection of microbubbles in patients with breast cancer. SUBJECTS AND METHODS: Eighty consecutive consenting patients with primary breast cancer were recruited. Patients received a periareolar intradermal injection of microbubble contrast agent. Breast lymphatics were visualized by sonography and followed to the axilla to identify SLNs. A guidewire was deployed to localize the SLN. The next day, patients underwent standard tumor excision and SLN biopsy. RESULTS: In 71 (89%) of the 80 patients, SLNs were identified and guidewires were inserted. In these patients, operative findings using conventional radioisotope and blue dye techniques confirmed that the wired nodes were SLNs. Fourteen patients were found to have metastases in SLNs. In these patients, the SLNs were identified correctly and were localized with guidewires before surgery. CONCLUSION: SLNs may be identified and localized before surgery using contrast-enhanced sonography after injection of microbubbles.