Potent Efficacy of 3-Amino-4-hydroxy Benzoic Acid, a Small Molecule Having Anti-fibrotic Activity, in a Mouse Model of Non-alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH), which is on the rise due to the increasing obese population and changing lifestyles, causes fibrosis over time and carries the risk of progression to cirrhosis and hepatocellular carcinoma. However, there are no approved effective treatments for NASH. Recent studies suggest that increased lipid metabolism and reduced nitric oxide content are responsible for NASH; 3-amino-4-hydroxy benzoic acid (AHBA) was identified as an inhibitor for the phosphatase activity of soluble epoxy hydrolase, which in turn inhibits lipid metabolism and endothelial nitric oxide synthase activity. The aim of this study was to assess the efficacy of AHBA in a mouse model of NASH. NASH was induced in mice by streptozotocin administration and a high-fat diet loading. The efficacy of AHBA was determined by measuring liver function using serum and liver samples and conducting a morphological assessment. AHBA considerably attenuated the increase in the liver weight and alkaline phosphatase content, which occurred due to the progression of NASH. Hepatocellular steatosis, inflammatory cell infiltration, and hepatocellular ballooning of hepatocytes remained unaltered. In contrast, AHBA treatment significantly ameliorated the fibrotic alterations within liver tissue that were induced by the onset of NASH. These results demonstrate the potential of AHBA as a therapeutic pharmaceutical compound that can treat NASH.

Inhibition of neuroinflammation by MIF inhibitor 3-({[4-(4-methoxyphenyl)-6-methyl-2-pyrimidinyl]thio}1methyl)benzoic acid (Z-312).

Microglial overactivation-mediated neuroinflammation contributes greatly to the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that is involved in the pathophysiology of various inflammatory diseases by inducing various proinflammatory cytokines. Compound 3-({[4-(4-methoxyphenyl)-6-methyl-2-pyrimidinyl]thio}methyl)benzoic acid (Z-312) is a novel small -molecule inhibitor of MIF tautomeric activity. In this study, we investigated the anti-inflammatory effects of Z-312 on liposaccharide (LPS)-induced neuroinflammation in vitro and in vivo. The results showed that Z-312 significantly decreased the production of nitric oxide (NO), interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-6 in LPS-stimulated microglial cells. Mechanistically, nuclear translocation of the p65 subunit of nuclear factor (NF)-κB, degradation and phosphorylation of IκBα, NF-κB transcriptional activity and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and JNK were markedly attenuated by pretreatment with Z-312 in BV-2 microglial cells. In addition, Z-312 suppressed the neurotoxic effects of cell culture medium of LPS-activated BV-2 microglia on cocultured mouse HT22 neuroblastoma cells. An in vivo study demonstrated that Z-312 markedly ameliorated microglial activation and subsequent DA neuron loss in an LPS-induced Parkinson's disease (PD) mouse model. These results suggest that MIF inhibitor Z-312 may be a promising neuroprotective agent for the treatment of neuroinflammation-mediated neurological diseases.

Dynamics-Based Discovery of Novel, Potent Benzoic Acid Derivatives as Orally Bioavailable Selective Estrogen Receptor Degraders for ERα+ Breast Cancer.

The estrogen receptor α (ERα) is identified as an effective target for the treatment of ERα+ breast cancer; thus, discovery of novel selective estrogen receptor degraders (SERDs) are developed as an effective method to overcome the resistance of breast cancer. Herein, the hot-spot residues for protein-ligand interaction between SERDs and ERα are analyzed by molecular dynamic simulation technology, focusing on the hot-spot residues for four series of designed and synthesized SERDs. SAR studies revealed that while the acrylic acid moiety of AZD9496 is scaffold hopping into benzoic acid, compound D24 exhibits potent binding affinity with ERα, good degradation efficacy of ERα, and inhibitory effect against the MCF-7 breast cancer cell line. Besides, D24 also displays good antitumor efficacy in the MCF-7 human breast cancer xenograft model in vivo, favorable pharmacokinetic properties, excellent druggability, and good safety property, making D24 as a promising drug candidate of SERD for further evaluation.

Design, synthesis and evaluation of 3-amide-5-aryl benzoic acid derivatives as novel P2Y14R antagonists with potential high efficiency against acute gouty arthritis.

P2Y14 nucleotide receptor plays important roles in series of physiological and pathologic events especially associated with immune and inflammation. Based on the 3-amide benzoic acid scaffold reported by our group previously, a series of 5-aryl-3-amide benzoic acid derivatives were designed as novel P2Y14 antagonists with improved pharmacokinetic properties. Among which compound 11m showed most potent P2Y14 antagonizing activity with an IC50 value of 2.18 nM, furnishing greatly improved water solubility and bioavailability compared with PPTN. In MSU-induced acute gouty arthritis model in mice, 11m exerted promising in vivo efficacy in alleviating mice paw swelling and inflammatory infiltration. Mechanistically, compound 11m notably blocked pyroptosis of macrophages through inhibiting NLRP3 inflammasome activation. This work may contribute to the identification of potential therapeutic agents to intervene in acute gouty arthritis.

Hydroxyl substituted benzoic acid/cinnamic acid derivatives: Tyrosinase inhibitory kinetics, anti-melanogenic activity and molecular docking studies.

The inhibition of tyrosinase is an established strategy for treating hyperpigmentation. Our previous findings demonstrated that cinnamic acid and benzoic acid scaffolds can be effective tyrosinase inhibitors with low toxicity. The hydroxyl substituted benzoic and cinnamic acid moieties of these precursors were incorporated into new chemotypes that displayed in vitro inhibitory effect against mushroom tyrosinase. The most active compound, (2-(3-methoxyphenoxy)-2-oxoethyl (E)-3-(4-hydroxyphenyl) acrylate) 6c, inhibited tyrosinase with an IC50 of 5.7 µM, while (2-(3-methoxyphenoxy)-2-oxoethyl 2, 4-dihydroxybenzoate) 4d had an IC50 of 23.8 µM. In comparison, the positive control, kojic acid showed tyrosinase inhibition with an IC50 = 16.7 µM. Analysis of enzyme kinetics revealed that 6c and 4d displayed noncompetitive reversible inhibition of the second tyrosinase enzymatic reaction with Ki values of 11 µM and 130 µM respectively. In silico docking studies with mushroom tyrosinase (PDB ID 2Y9X) predicted possible binding modes in the catalytic site for these active compounds. The phenolic para-hydroxy group of the most active compound 6c is predicted to interact with the catalytic site Cu++ ion. The methoxy part of this compound is predicted to form a hydrogen bond with Arg 268. Compound 6c had no observable toxic effects on cell morphology or cell viability at the highest tested concentration of 91.4 µM. When dosed at 91.4 µM onto B16F10 melanoma cells in vitro6c showed anti-melanogenic effects equivalent to kojic acid at 880 µM. 6c displayed no PAINS (pan-assay interference compounds) alerts. Our results show that compound 6c is a more potent tyrosinase inhibitor than kojic acid and is a candidate for further development. Our exposition of the details of the interactions between 6c and the catalytic pocket of tyrosinase provides a basis for rational design of additional potent inhibitors of tyrosinase, built on the cinnamic acid scaffold.

Benzoic Acid Used as Food and Feed Additives Can Regulate Gut Functions.

As a kind of antibacterial and antifungal preservative, benzoic acid is widely used in foods and feeds. Recently, many studies showed that it could improve the growth and health, which should, at least partially, be derived from the promotion of gut functions, including digestion, absorption, and barrier. Based on the similarity of gut physiology between human and pigs, many relative studies in which piglets and porcine intestinal epithelial cells were used as the models have been done. And the results showed that using appropriate benzoic acid levels might improve gut functions via regulating enzyme activity, redox status, immunity, and microbiota, but excess administration would lead to the damage of gut health through redox status. However, the further mechanisms that some intestinal physiological functions might be regulated are not well understood. The present review will, in detail, summarize the effect of benzoic acid on gut functions.

Benzoic acid in nursery diets increases the performance from weaning to finishing by reducing diarrhoea and improving the intestinal morphology of piglets inoculated with Escherichia coli K88.

A total of 224 weaned pigs (DanBred sows x PIC 337 sires) with an average body weight (BW) of 6.37 ± 0.34 kg (21 days of age) were used to evaluate how different levels of benzoic acid fed to weaning pigs orally inoculated with Escherichia coli (K88+ ) affected the nursing and grow-finishing performance, the physicochemical properties of the intestine, the volatile fatty acid concentration in the caecum and the incidence of diarrhoea. Pigs were randomly allocated in an experimental design of randomized blocks in a 4 × 2 factorial design, and they were administered four levels of benzoic acid (0.00%, 0.25%, 0.50% and 0.75%) and inoculated (or not) in two consecutive days with 1 ml solution containing 106 CFU/ml of E. coli (K88+ ). Seven replicates (pens) per treatment were used, and four animals were kept per pen. Supplementation with 0.75% benzoic acid promoted better performance (p < 0.05) in the nursery phase as well as in the subsequent phases until slaughter, and it decreased the incidence of diarrhoea in piglets (p < 0.05). In the piglets fed the benzoic acid diet, the villus height in the jejunum and ileum was greater until 42 days of life (p < 0.05), the crypt depth was decreased in the caecum (p < 0.05), and the butyric acid concentration was increased in the caecal content tendencially (p = 0.0708). In conclusion, supplementation with 0.75% benzoic acid has a positive effect on piglets by reducing diarrhoea, improving intestinal health and promoting the performance from weaning to finishing. Thus, benzoic acid can be considered a potential alternative that can replace growth-promoting antibiotics.

OUR EXPERIENCE WITH THE USE OF 40% BENZOIC ACID FOR NECRECTOMY IN DEEP BURNS.

INTRODUCTION: Chemical necrectomy of deep burns using 40% benzoic acid has been used extensively by the Department of Burns and Reconstructive Surgery at the University Hospital since its establishment in 1982. In spite of definite advantages for the patient and medical staff, hard data concerning benzoic acid absorption through skin necrosis and patient safety was missing. MATERIAL AND METHODS: We examined 22 burn patients in collaboration with the University Hospital Brno, Department of Clinical Biochemistry. The plasmatic levels of benzoic acid, hippuric acids and glycine, which is consumed during the metabolism of benzoic acid, were measured. Urine samples were collected to determine the total amount of hippuric acid that is excreted. We were able to determine the total amount of absorbed and excreted benzoic acid from these values. RESULTS: We consistently found that there was a rapid and short-term increase of plasmatic levels of benzoic acid (maximum 1.3 mmol/l). This value is about 5 times lower than the minimum toxic level of this acid (6.5 mmol/l). The same course has been observed in hippuric acid. The level of glycine dropped slightly, but was still within the normal range. DISCUSSION: Typical and atypical courses of the levels of both acids were discussed as well as the correlation of the dynamics of elimination with the extent of benzoic acid application in relationship with the clinical status of the patient. The effectiveness and safety of this method was evaluated. CONCLUSION: After summarizing the observations, it was demonstrated that chemical necrectomy using 40% benzoic acid is a selective method comparable with other types of sharp necrectomy. Chemical necrectomy is inexpensive, easy to perform and also reduces blood loss. Toxicity of absorbed benzoic acid is clinically negligible. Furthermore, benzoic acids antimycotic and antibacterial properties prevent the development of wound infection.

CLINICAL MANAGEMENT OF BILATERAL CUTANEOUS SQUAMOUS CELL CARCINOMA OF THE HIND FEET PADS IN A SOUTHERN WHITE RHINOCEROS (CERATOTHERIUM SIMUM SIMUM).

The current report describes the temporary regression, due to intensive symptomatic treatment, of ulcerative skin lesions caused by squamous cell carcinoma in a white rhinoceros. A captive, 40-yr-old southern white rhinoceros (Ceratotherium simum simum) developed profound, ulcerative skin lesions on the pads of both hind feet. At the peak of the disease, at least one quarter of the pads was affected. A diagnosis of squamous cell carcinoma was made via biopsy. Treatment included anti-inflammatory drugs, antibiotics, and local care. The lesions regressed on both feet until they seemed clinically healed. It was presumed that long-term, anti-inflammatory treatment and local bandaging had induced the temporary regression of the lesions. Two years later, however, a small ulcerative lesion reappeared on one pad and post mortem examination confirmed that the carcinoma was also histologically present in the clinically intact tissue. No metastasis was found and computed tomography showed normal digital bones.

Tiña negra: un reporte en Matanzas, Cuba / Tinea nigra: a report in Matanzas, Cuba

La tiña negra es una micosis cutánea superficial causada por un hongo levaduriforme denominado Hortaea werneckii, es frecuente en regiones cálidas y en zonas de playa. Se comunica su aislamiento de la palma de la mano de un niño sin antecedentes de contacto con fuente infecciosa ni de hiperhidrosis que fue tratado con pomada de Whitfield con azufre desapareciendo la lesión al mes sin recidivas. Se considera significativo presentar este caso dada la escasa frecuencia de su diagnóstico(AU)

Tinea nigra is a superficial cutaneous mycosis, caused by the yeasty-form fungus called Hortaea werneckii. It is frequent in warm regions and in beach zones. Its isolation in the hand palm of a boy without antecedents of infectious contact or hyperhidrosis is reported in this article. He was treated with an ointment of benzoic acid, salicylic acid and sulfur; lesions disappeared after a month without recurrence. The presentation of the case was considered significant due to the scarce frequency of this diagnose(AU)

Discovery of potent aryl-substituted 3-[(3-methylpyridine-2-carbonyl) amino]-2,4-dimethyl-benzoic acid EP4 antagonists with improved pharmacokinetic profile.

Two new series of EP4 antagonists containing a 3-methylaryl-2-carbonyl core have been identified. One series has a 3-substituted-phenyl core, while the other one incorporates a 3-substituted pyridine. Both series led to compounds with potent activity in functional and human whole blood (hWB) assays. In the pyridine series, compound 7a was found to be a highly potent and selective EP4 antagonist, with suitable rat and dog pharmacokinetic profiles.

New cyclopentaquinoline derivatives with fluorobenzoic acid induce G1 arrest and apoptosis in human lung adenocarcinoma cells.

Non-small cell lung cancer accounts for 80-85% of all lung cancer cases and is the leading cause of cancer death indicating inefficient current treatment. Acridine derivatives interact with DNA and inhibit topoisomerase leading to cell growth arrest or cell death. The aim of this study was to evaluate the effects of new synthesized sixteen 2,3-dihydro-1H-cyclopenta[b]quinoline derivatives (cyclopentaquinoline), a member of acridine-based compounds, on the survival and growth of human lung adenocarcinoma, A549 cells. Anticancer activity of eight new cyclopentaquinoline derivatives with hydrazinonicotinic acid (compounds 1-8) and eight with fluorobenzoic acid (compounds 9-16) were screened using WST-1 assay. Interestingly, cyclopentaquinoline derivatives with fluorobenzoic moiety were found to have a higher anticancer activity than derivatives with hydrazinonicotinic acid. Four out of eight tested compounds with fluorobenzoic acid inhibited 50% cancer cell growth at concentration below 20µM. Moreover, the efficacy of cyclopentaquinoline derivatives containing fluorobenzoic acid correlated with increasing number of carbon atoms in the aliphatic chain. The most effective compounds (6, 15, 16) were selected to determine molecular mechanisms of their anticancer action. The results indicated that inhibition of A549 cell growth by compounds 15 and 16 was associated with a cell cycle arrest at G0/1 phase and with induction of caspase 3-dependent apoptosis. Compound 6 also caused A549 cells death due to apoptosis, however, it had no significant effect on a cell cycle progression. These findings suggest that cyclopentaquinoline derivatives containing fluorobenzoic acid with 8 and 9 carbon atoms in aliphatic chain may be promising candidate for treatment of lung cancer.

A series of D-amino acid oxidase inhibitors specifically prevents and reverses formalin-induced tonic pain in rats.

We have found that mutation of D-amino acid oxidase (DAO) diminished formalin-induced tonic pain. The present research further studied the analgesic effects of a series of DAO inhibitors in this model. 5-Chlorobenzo[d]isoxazol-3-ol (CBIO), 4H-thieno[3,2-b]pyrrole-5-carboxylic acid (compound 8), 5-methylpyrazole-3-carboxylic acid (AS057278), sodium benzoate, and 4-nitro-3-pyrazole carboxylic acid (NPCA) inhibited rat spinal cord-derived DAO activity in a concentration-dependent manner, with maximal inhibition of 100% and potency rank of CBIO > compound 8 > AS057278 > sodium benzoate > NPCA. In rats, intrathecal injections of CBIO, compound 8, AS057278, and sodium benzoate but not NPCA specifically prevented formalin-induced tonic pain but not acute nociception, with the same potency order as in the DAO activity assay. The highly potent analgesia of DAO inhibitors was evidenced by CBIO, which prevented 50% pain at 0.06 µg, approximately 5-fold the potency of morphine. CBIO given after formalin challenge also reversed the established pain state to the same degree as prevention. The antihyperalgesic potencies of these DAO inhibitors were highly correlated to their inhibitions of spinal DAO activity. Maximum inhibition of pain by these compounds was approximately 60%, comparable with that of the N-methyl-D-aspartic acid receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), suggesting that a larger portion of formalin-induced tonic pain is "DAO-sensitive," whereas the remaining 40% of tonic pain and acute nociception is "DAO-insensitive." These findings, combined with our previous DAO gene mutation and induction results, indicate spinal DAO mediates both induction and maintenance of formalin-induced tonic pain and further validate spinal DAO as a novel and efficacious target molecule for the treatment of chronic pain.

Comparative effects of various commercially available mouthrinse formulations on oral malodor.

OBJECTIVES: The primary aim of this study was to compare a new mouthwash (SB12®) containing 0.025% chlorhexidine and 0.3% zinc for oral malodor reduction against four commercially available mouthwashes and negative control. A secondary aim was to compare the two methods for measuring volatile sulphur compounds (VSC) by halimetry and OralChroma. METHODS: Organoleptic scale, halimeter and the OralChroma were used to assess oral malodour and VSC. The effects of five test formulations and water (negative control) were assessed after 30, 60, 90 and 180 min, with 1 week between the treatments to avoid any cross-over effect. RESULTS: Reduction in H(2) S by halimetry and malodour levels by organoleptic assessment ranged from, slight (LacerFresh®) (P > 0.05), moderate (BreathRx®, SmartMouth® (P < 0.01) to marked effects (SB12®, Listerine®) (P < 0.001) at all time points compared with water. The largest differences were observed at 30 min and decreased with time. SB12® showed separation from Listerine® at 180 min, using ANOVA plus Bonferroni's Multiple Comparison post-test (P < 0.05). Relationships between organoleptic, halimeter and OralChroma were between R² = 0.795 and 0.926. CONCLUSION: SB12 shows a consistent and reproducible inhibitory effect on oral malodor parameters, which in turn correlate well with each other.

A novel Ca2+/calmodulin antagonist HBC inhibits angiogenesis and down-regulates hypoxia-inducible factor.

Recent reports have shown that Ca(2+)/calmodulin (Ca(2+)/CaM) signaling plays a crucial role in angiogenesis. We previously developed a new Ca(2+)/CaM antagonist, HBC (4-{3,5-bis-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-4,5-dihydropyrazol-1-yl}benzoic acid), from a curcumin-based synthetic chemical library. Here, we investigated its anti-angiogenic activity and mode of action. HBC potently inhibited the proliferation of human umbilical vascular endothelial cells with no cytotoxicity. Furthermore, HBC blocked in vitro characteristics of angiogenesis such as tube formation and chemoinvasion, as well as neovascularization of the chorioallantoic membrane of growing chick embryos in vivo. Notably, HBC markedly inhibited expression of hypoxia-inducible factor-1alpha (HIF-1alpha) at the translational level during hypoxia, thereby reducing HIF-1 transcriptional activity and expression of its major target gene, vascular endothelial growth factor. In addition, combination treatment with HBC and various HIF-1 inhibitors, including suberoylanilide hydroxamic acid, rapamycin, and terpestacin, had greater anti-angiogenic activity than treatment with each single agent. Collectively, our findings indicate that HBC is a new anti-angiogenic agent targeting HIF that can be used to explore the biological role of Ca(2+)/CaM in angiogenesis.

2-hydroxy 4-methoxy benzoic acid isolated from roots of Hemidesmus indicus ameliorates liver, kidney and pancreas injury due to streptozotocin-induced diabetes in rats.

Protective effect was evaluated in streptozoticin (STZ)-induced diabetes rats. 2-Hydroxy 4-methoxy benzoic acid (HMBA) was isolated from the roots of Hemidesmus indicus and administered (500 microg/kg body weight) orally for 7 weeks to STZ-induced diabetic and non-diabetic rats to study its effect on protein metabolism, serum electrolytes and on liver and kidney lipid peroxides. Oral administration of HMBA restored the altered biochemical parameters such as urea, uric acid, creatinine, plasma proteins and serum electrolytes to near-normal levels. HMBA treatment significantly decreased lipid peroxidation and malondialdehyde levels in diabetic liver and kidney. Effect of HMBA was equivalent to that of the standard drug, tolbutamide (100 mg/kg body wt). The histological changes were also in correlation with the biochemical findings. The present study showed that HMBA isolated from H. indicus roots had ameliorative effect on liver, kidney and pancreatic injury in STZ-induced diabetic rats.

Determination of benzoic acid in serum or plasma by gas chromatography-mass spectrometry (GC/MS).

Nonketotic hyperglycinemia (NKH), a metabolic disorder due to defects in the glycine cleavage system, leads to the accumulation of toxic levels of glycine. Glycine levels in these patients may be lowered by sodium benzoate treatment. Benzoic acid binds to glycine to form hippurate, which is subsequently eliminated through the kidneys. At high concentrations, hippuric acid can crystallize in the kidneys and cause renal failure. Therefore, it is desirable to have benzoic acids concentrations within a therapeutic range. In the gas chromatography method described, the drug from the acidified serum or plasma sample is extracted using ethyl acetate. The organic phase containing drug is separated and dried under a stream of nitrogen. After trimethylsilyl derivatization, benzoic acid analysis is done on a gas chromatograph mass spectrometer. Quantitation of the drug in a sample is achieved by comparing responses of the unknown sample to the responses of the calibrators using selected ion monitoring. Benzoic acid D(5) is used as an internal standard.

Resorption and elimination kinetics of benzoic acid during chemical necrectomy in deep burns.

OBJECTIVE: Chemical necrectomy is an alternative to the surgical or sharp necrectomy for the removal of necrotic parts of the skin in the treatment of deep burns. The aim of our work was to monitor the dynamics of resorption and elimination of benzoic acid applied to the burnt skin. METHODS: The set consisted of 10 patients (9 men; 1 woman) aged 25-57 years with IIb-III-degree skin burns. 40% benzoic acid in white petrolatum was applied to the burnt area to the extent of 3-5% of TBSA (total body surface area) for a period of 48 hours. The concentrations of benzoic acid, hippuric acid, and glycine in the serum was monitored at the 10th, 20th, 60th, 120th, 240 th and 360 th minute thereafter and further at the 12th, 24th, 48th, and 72nd hour; the excretion of hippuric acid in urine was monitored in six 12-hour intervals. RESULTS: The highest concentration of benzoic acid in the serum was detected in the 60th minute sample (0.094+/-0.074 mmol/L) and of hippuric acid in the 120th minute sample (0.234+/-0.088 mmol/L) from the application of benzoic acid to the burnt skin. In the period between the 6th and 48th hour, the average concentration of benzoic acid in the serum ranged between 0.042 and 0.03 mmol/L. In this period there was also a significant decrease in serum glycine concentration (p<0.05). During the 48-hour application of benzoic acid to the burnt skin, 46.0-145 mmol of hippuric acid was excreted in urine. CONCLUSION: Chemical necrectomy with the use of 40% benzoic acid led only to a moderate increase of its concentration in the serum. After its resorption from the wound area it is transformed to hippuric acid, which is promptly excreted in urine.

Deficiency of the carnitine transporter (OCTN2) with partial N-acetylglutamate synthase (NAGS) deficiency.

A patient with recurrent episodes of hyperammonaemia (highest ammonia level recorded 229 micromol/L, normal 9-33) leading to altered levels of consciousness was diagnosed with partial N-acetylglutamate synthase (NAGS) deficiency (9% residual activity) at age 5 years and was treated with ammonia-conjugating agents (Ucephan 250 mg/kg per day and later sodium phenylbutyrate 200-250 mg/kg per day) for 15 years. A chronically low serum carnitine level (pretreatment plasma free carnitine 4 nmol/L, normal 37 +/- 8 nmol/L; total carnitine 8 nmol/L, normal 46 +/- 10) was assumed to be secondary and was treated with supplemental carnitine (30-50 mg/kg per day). Hypoglycaemia (blood sugar 35 mg/dl, normal 70-100), cardiomegaly, and fatty liver were also noted at diagnosis. The patient died unexpectedly at age 20 years. In retrospect, it was learned that the patient had stopped his carnitine without medical consultation several weeks prior to his death. Additional molecular investigations identified two mutations (R254X and IVS3 + 1G > A) in the patient's OCTN2 (SLC22A5) gene, consistent with a diagnosis of primary carnitine deficiency due to carnitine transporter defect. R245X is a founder mutation in Southern Chinese populations. It is unknown whether the original NAGS deficiency was primary or secondary, but molecular analysis of the NAGS gene failed to identify mutations. Urea cycle enzyme expression may be affected by fatty acid suppression of an AP-1 binding site in the promoter enhancer region of the urea cycle gene. Regardless, it is clear that the NAGS abnormality has led to delay of recognition of the OCTN2 defect, and modified the clinical course in this patient.

Benzoate treatment and the glycine index in nonketotic hyperglycinaemia.

High-dose benzoate treatment aimed at reducing plasma glycine levels to normal reduces seizures and increases wakefulness in patients with nonketotic hyperglycinaemia (NKH). Since benzoate metabolism is dependent on the available glycine pool, and since the glycine pool is variably affected by the deficiency in the glycine cleavage enzyme system, we examined the importance of interpatient variability in benzoate requirement. To correct for the dietary glycine contribution, the glycine index was introduced as the molar requirement of benzoate dose necessary to normalize plasma glycine levels and subtracting from that the dietary glycine intake, both corrected for weight. The glycine index varied between 3.62 and 4.87 mmol/kg per day in five patients with a poor neurodevelopmental outcome and between 0.92 and 1.90 mmol/kg per day in four patients with a better neurodevelopmental outcome, and was 2.54 mmol/kg per day in a single patient with an intermediate outcome. The glycine index was stable over time within each patient. Exceeding the balance by either increasing food glycine intake or decreasing the benzoate dose resulted in increased glycine levels. Exceeding the glycine tolerance by increasing benzoate resulted in elevated and toxic levels of benzoate. The glycine index is a stable, individually specific parameter in patients with NKH. It has clinical consequences for the dose of benzoate required and the role of dietary management. Through its correlation with neurodevelopmental outcome, the glycine index points to potential genetic factors that could contribute to the psychomotor retardation in NKH.