Ethnic differences in GRHPR mutations in patients with primary hyperoxaluria type 2.

The objective of this study was to investigate ethnic differences in the glyoxylate reductase/hydroxypyruvate reductase (GRHPR) gene in patients with primary hyperoxaluria type 2 (PH2). GRHPR was genotyped in Japanese patients with PH2 and all GRHPR mutations described to date were reviewed in terms of geographic and ethnic association. We identified a novel mutation, a two-nucleotide deletion (c.248_249delTG) in exon 3 creating a premature 'stop' at codon 91. Also, we found that the c.864_865delTG mutation was associated with the rs35891798 single-nucleotide polymorphism. The allelic frequencies of the c.103delG, c.494G>A, c.403_404+2 delAAGT, and c.864_865delTG mutations in PH2 patients were 37.8%, 15.6%, 10.0%, and 10.0%, respectively. All patients with the c.103delG mutation were Caucasian. Patients with the c.494G>A mutation and 78% (7/9) of those with the c.403_404+2 delAAGT mutation were from the Indian subcontinent, whereas those with the c.864_865delTG mutation were Chinese or Japanese. Molecular analysis of GRHPR of four Japanese PH2 patients identified a novel mutation (c.248_249delTG in exon 3). Caucasians with PH2 should be screened for the c.103delG mutation; patients from the Indian subcontinent for c.494G>A; and patients of East Asian origin (particularly) for c.864_865delTG. The prevalence of the latter mutation in PH2 patients from East Asia was 75.0%.

Disseminated mucocutaneous herpes simplex virus infection in an immunocompetent woman.

Disseminated mucocutaneous herpes simplex virus (HSV) infection in an immunocompetent person is quite rare. A 19-year-old healthy Japanese woman presented with painful, umbilicated vesicles and pustules on her genital region, both nipples and on the forearm 10 days after the last sexual contact with her partner who had cold sore at that time. Tzanck test and biopsy confirmed the diagnosis of disseminated mucocutaneous HSV infection. She did not have any visceral HSV disease. Skin lesions improved after treatment with acyclovir and erythromycin for seven days. We propose that like herpes gladiatorum, HSV dissemination in this case was acquired by close body contact.

Herpes zoster of the nipple: rapid DNA-based diagnosis by the loop-mediated isothermal amplification method.

A 28-year-old Japanese man presented with grouped erosions and vesicles on an erythematous base affecting the right areola and the surrounding skin. A Tzanck smear from the vesicle revealed giant cells. An initial clinical diagnosis of mammary herpes simplex was considered but to explore the differential diagnosis, viral DNA was amplified by the loop-mediated isothermal amplification (LAMP) method. DNA replication was observed only in varicella zoster virus LAMP mixture, and this confirmed a diagnosis of herpes zoster. The patient was treated with 3000 mg of daily oral valacyclovir for seven days. After antiviral treatment, the lesion had healed and the pain had resolved completely.

Five cases of beta-ureidopropionase deficiency detected by GC/MS analysis of urine metabolome.

The clinical presentation of inborn errors of pyrimidine degradation varies considerably from asymptomatic to severe neurological illness. We have reported a method to screen for and make a chemical diagnosis of beta-ureidopropionase deficiency, leading to the discovery of the first asymptomatic case of this disease. In this method, the recovery of beta-ureidopropionate and beta-ureidoisobutyrate, the key biomarkers, was very high,and the adoption of GC/MS and targeted analysis enabled us to simultaneously obtain information related and unrelated to pyrimidine metabolism. The present study reports the results of a large-scale screening of 24,000 newborns using dried urine on filter paper. Identification of a total of four asymptomatic patients among newborns suggests the high incidence (1/6000) of this disease in Japan. While these newborns were asymptomatic, two additional cases detected at the age of 5 years as well as 3 months with this method for high-risk screening had autism and West syndrome, respectively.The key biomarkers and alpha-ureidobutyrate used as an internal standard were found to give not only their di-trimethylsilyl derivatives but also tri-trimethylsilyl derivatives, upon derivatization. The mass spectra and retention times of their tri-trimethylsilyl derivatives and data handling for quantification of the markers are presented.Identification of individuals with defects in pyrimidine metabolism would realize personalized medication in cancer chemotherapy with pyrimidine analogs such as 5-fluorouracil.

A pilot study to assess the efficacy of photodynamic therapy for Japanese patients with actinic keratosis in relation to lesion size and histological severity.

BACKGROUND/PURPOSE: Topical 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) is effective for actinic keratosis (AK); few studies have examined Oriental patients. The aim of this study is to assess the efficacy of PDT for the treatment of Japanese AK patients classified by lesion size and histological severity. METHODS: Thirty patients with solitary AK lesions were divided into two groups according to diameter: a small lesion group (SL), diameter < or =10 mm and a larger lesion group (LL), diameter >10 mm, and histological severity: Group I (mild and moderate) and Group II (severe). After application of 20% ALA for 4 h, exposure to an excimer-dye laser at 630 nm was performed at a dose of 50 J/cm(2) three times at an interval of 7 days. Therapeutic effects were assessed and followed for 12 months. RESULTS: In all 10 SL patients, atypical cells disappeared after PDT and did not recur for 12 months. However, for the 20 LL patients, recurrence was seen in 2 of the 14 Group I patients, while 4 of 6 Group II patients showed residual tumor cells after the first PDT session. CONCLUSION: The present study demonstrated that ALA-PDT might be useful for treatment of Japanese AK. The therapeutic outcome might depend on the lesion size and the histopathological severity.

Etretinate enhances the susceptibility of human skin squamous cell carcinoma cells to 5-aminolaevulic acid-based photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (5-ALA) is a noninvasive and effective treatment for superficial skin cancers. Etretinate, a derivate of vitamin A, with the chemical formula ethyl(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nona-tetraenoate, has been reported to have antitumour effects and to regulate the proliferation and differentiation of skin cancers. OBJECTIVE: In order to develop more efficient PDT, we investigated whether etretinate enhanced the cytotoxic action of ALA-based PDT against human squamous cell carcinoma cell line, HSC-5. METHOD: The in vitro cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic cells were detected by double-staining with fluorescent annexin V and propidium iodide. Intracellular protoporphyrin IX (PpIX) converted from exogenous ALA was measured by a fluorescence meter. RESULTS: HSC-5 cells pretreated with a nontoxic concentration of etretinate became more susceptible to the cytotoxic action of ALA-based PDT. Etretinate-pretreated cells underwent apoptosis in response to ALA-based PDT. Etretinate pretreatment resulted in enhanced accumulation of ALA-dependent intracellular PpIX. CONCLUSIONS: The results suggest that etretinate enhances the susceptibility of HSC-5 cells to ALA-based PDT via the intracellular increase of ALA-dependent PpIX. Etretinate might be useful for improvement of ALA-based PDT.

Effects of Bifidobacterium breve A1 on the cognitive function of older adults with memory complaints: a randomised, double-blind, placebo-controlled trial.

In our previous study, we reported the therapeutic potential of Bifidobacterium breve A1 in preventing cognitive impairment in a mouse model of Alzheimer's disease and participants with mild cognitive impairment; we suggested that probiotic supplementation is an effective therapeutic strategy for managing cognitive function. Accordingly, we conducted a randomised, double-blind, placebo-controlled trial to assess whether 12-week B. breve A1 supplementation could affect the cognitive function of elderly subjects with memory complaints. We assessed cognitive function using the Japanese version of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and Mini-Mental State Examination (MMSE) at baseline and after 12 weeks of probiotic supplementation. A total of 121 participants were randomised and received B. breve A1 capsules or placebo daily for 12 weeks; of these, 117 participants completed the study. At 12 weeks, neuropsychological test scores significantly increased in both groups; no significant intergroup difference was observed in terms of changes in scores from the baseline scores. However, a stratified analysis revealed a significant difference between B. breve A1 and placebo groups in terms of the subscale 'immediate memory' of RBANS and MMSE total score in the subjects with low RBANS total score at baseline. No significant differences in terms of blood parameters between the groups or adverse effects caused by B. breve A1 intervention were observed. The results of the present study suggest the safety of B. breve A1 supplementation and its potential in maintaining cognitive function in elderly subjects with memory complaints. However, future large-scale studies on individuals with impaired cognitive function are required to validate the present findings.

Bifidobacterium breve MCC1274 with glycosidic activity enhances in vivo isoflavone bioavailability.

Polyphenols are plant derived compounds that exert many beneficial health effects to the human host. However, associated health benefits of dietary polyphenol are highly dependent on their intestinal metabolism, bioavailability, and absorption. Bifidobacteria, which represent the key members of gut microbiota, have been suggested to promote gut microbial homeostasis and may be involved in the metabolism of polyphenols. In this study, the capabilities of thirteen Bifidobacterium strains in hydrolysing polyphenol glycosides were evaluated. Among the tested strains, Bifidobacterium breve MCC1274 was found to possess the highest ß-glucosidase activity and strong capability to convert daidzin and trans-polydatin to their aglycones; while kinetic analysis revealed that B. breve MCC1274 hydrolysed more than 50% of daidzin and trans-polydatin at less than 3 h of incubation. Further investigation using rats with an antibiotics-disturbed microbiome revealed that following the ingestion of daidzin glycoside, oral administration of B. breve MCC1274 significantly enhanced the plasma concentration of daidzein in rats pre-treated with antibiotics as compared to antibiotics-pre-treated control and non-treated control groups. The relative abundance of Actinobacteria and the total numbers of B. breve were also significantly higher in antibiotics-pre-treated rats administered with B. breve MCC1274 than that of the control groups. These findings suggest that B. breve MCC1274 is effective in enhancing the bioavailability of daidzein in the gut under dysbiosis conditions and may potentially improve intestinal absorption of isoflavones and promote human health.

Inhibitory effects of bovine lactoferrin on colon carcinoma 26 lung metastasis in mice.

In order to determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF), and related compounds on tumor growth and metastasis, bovine LF (bLF), and bLF hydrolysate and lactoferricin (bLFcin), active products generated by acid-pepsin hydrolysis were administered orally to BALB/c mice bearing subcutaneous (s.c.) implants of the highly metastatic colon carcinoma 26 (Co 26Lu). bLF and the bLF hydrolysate demonstrated significant inhibition of lung metastatic colony formation from s.c. implanted tumors without appreciable effects on tumor growth. bLFcin displayed a tendency for inhibition of lung metastasis. On the other hand, bLF did not exert marked anti-metastatic activity in athymic nude mice bearing Co 26Lu, though bLF had a tendency to inhibit the lung metastatic colony formation associated with anti-asialoGM1 antibody (Ab) treatment. AsialoGM1+ and CD8+ cells in white blood cells were increased after treatment with bLF. In vitro, the viability of Co 26Lu-F55 cells was markedly decreased when co-cultured with white blood cells from mice administrated bLF p.o., but recovered on treatment with anti-asialoGM1 Ab or anti-CD8 mAb and complement. The results suggest bLF and related compounds might find application as tools in the control of metastasis and that asialoGM1+ and CD8+ cells in the blood are important for their inhibitory effects.

Mitochondrial trifunctional protein deficiency associated with recurrent myoglobinuria in adolescence.

A 23-year-old man with recurrent myoglobinuria had low muscle-free carnitine levels and deficient fasting ketogenesis. Urinary organic acid analysis showed large amounts of C6-C14 3-hydroxydicarboxylic acids. Mitochondrial trifunctional protein (TP), harboring long-chain enoyl-coenzyme A (CoA) hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase showed markedly decreased activity in fibroblasts. On immunoblot analysis, the TP content of his fibroblasts was less than 2% that of the control cells. TP deficiency can be a life-threatening disorder with early infantile onset, but it can also present in adolescence with recurrent myoglobinuria.

Inhibitory effects of bovine lactoferrin on intestinal polyposis in the Apc(Min) mouse.

Chemopreventive effects of bovine lactoferrin (bLF), previously shown to strongly inhibit intestinal carcinogenesis in rats (K. Sekine, E. Watanabe, J. Nakamura, N. Takasuka, D.J. Kim, M. Asamoto, V. Krutovskikh, T.H. Baba, T. Ota, M.A. Moore, M. Masuda, H. Sugimoto, H. Nishino, T. Kakizoe, H. Tsuda, Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats, Jpn. J. Cancer Res. 88 (1997) 523-526; K. Sekine, Y. Ushida, T. Kuhara, M. Iigo, H. Baba-Toriyama, M.A. Moore, M. Murakoshi, Y. Satomi, H. Nishino, T. Kakizoe, H. Tsuda, Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane, Cancer Lett. 121 (1997) 211-216), on spontaneous intestinal polyp development were assessed in the ApcMin mouse, a model for both familial adenomatous polyposis and sporadic colon cancers. In the experiment, 54 mice at 6 weeks of age were given 2% bLF (15 mice), 0.2% bLF (15 mice) and AIN-93G (24 mice) as basal diet ad libitum for 8 weeks. An overall tendency for a reduction in the total number of polyps in the small intestine was evident in the bLF-treated animals, along with significant suppression in the jejunum at the 2% dose (P < 0.05, 68% of the control). In addition, body growth suppression, presumed to be due to anemia and/or intussusception as a consequence of numerous polyps in the intestine, was alleviated. No toxic effects were observed in the intestinal epithelium. Although not as obvious as observed for the rat case, the data suggest that bLF may be a chemopreventor of intestinal polyposis.

Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane.

The influence of concomitant administration of bovine lactoferrin (bLF) on induction of aberrant crypt foci (ACF) by azoxymethane was investigated in male F344 rats. Two percent bLF and 3% Bifidobacterium longum (B. longum), as a positive control, significantly decreased the numbers of ACF as well as the total numbers of aberrant crypts reproducibly in three independent studies (2% bLF, P < 0.01; 3% B. longum, P < 0.05). Most importantly large size foci composed of four or more crypts were always significantly decreased by 2% bLF (P < 0.05). Additional investigation of the natural killer activity of spleen cells demonstrated enhancement by bLF (P < 0.01) and B. longum (P < 0.01) in line with the levels of influence on foci induction, indicating a possible role for elevated immune cytotoxicity in the observed inhibition.

Ergosterol biosynthesis in yeast. Pathways in the late stages and their variation under various conditions.

[Methyl-14C]methionine was supplied to yeast cells under aerobic and anaerobic conditions for the investigation of the pathway for ergosterol biosynthesis after the methylation of the side-chain. Under aerobic conditions, the incorporation of radioactivity into ergosterol was high. With a limited oxygen supply, in contrast, the radioactivity was first accumulated in ergosta-7,24(28)-dien-3beta-ol and ergosta-8,24(28)-dien-3beta-ol, and then transferred to ergost-7-en-3beta-ol, ergost-8-en-3beta-ol and ergosta-7,22-dien-3beta-ol with time. Under strictly anaerobic conditions, a double bond was introduced neither to delta5 nor to delta22. The results of the tracer experiments suggested the operation of several pathways in the late stages of ergosterol biosynthesis. It was also suggested that the main pathways varied depending on the conditions such as oxygen supply and other factors. The above conclusion was supported by the results of the analyses of the sterol compositions of the cells grown under various conditions.

Increased excretion of lactate, glutarate, 3-hydroxyisovalerate and 3-methylglutaconate during clinical episodes of propionic acidemia.

Metabolic changes dependent upon clinical conditions were studied in an eight-month-old girl with propionyl CoA carboxylase deficiency. Only methylcitric acid and 2-methyl-3-oxovaleric acid were detected in the urine of the patient under clinically favorable conditions. During episodes of clinical decompensation, she excreted increased amounts of all the metabolites associated with this disorder. Four acetyl CoA precursors increased during clinical episodes: glutaric acid, a catabolic intermediate of lysine; 3-hydroxyisovaleric acid and 3-methylglutaconic acid, catabolic intermediates of leucine; and lactic acid. This suggests that under clinically favorable conditions the patient has an altered propionate metabolism which proceeds via normal acetyl CoA metabolism with sufficient capacity for acetyl CoA plus propionyl CoA metabolism. When the production of propionyl CoA exceeds the metabolic capacity, however, the catabolism of potent ketogenic amino acids is effectively suppressed in order to reduce acetyl CoA production.

Benzoylalanine: detection and identification of an alanine conjugate with benzoic acid in hyperammonemic patients treated with sodium benzoate.

Urine of four hyperammonemic patients who were treated with large amounts of sodium benzoate were analyzed. Gas chromatographic and gas chromatograph-mass spectrometric analysis showed the presence of large amounts of benzoylalanine in addition to much hippuric acid. Benzoylalanine was identified by comparison with an authentic standard by means of its mass spectrum and the gas chromatographic retention time of its trimethylsilyl derivative. This abnormal metabolite is thought to be derived from the conjugation of benzoyl CoA with alanine. It is suggested that the excretion of benzoylalanine results from a reduction in the liver free glycine level caused by hippuric acid synthesis.

Altered metabolic profiles of valproic acid in a patient with Reye's syndrome.

Urinary valproate metabolite and endogenous organic acid profiles in a 6-yr-old girl with Reye's syndrome were investigated by means of gas chromatography-mass spectrometry. 2-n-Propyl-3-oxovaleric acid, normally the major metabolite of valproate in man, was undetectable, while 2-n-propylglutaric acid, the end product via omega-oxidation was markedly increased. Polyunsaturated valproate was not found. Valproate-glucuronide was still found as the major metabolite. The clinical findings coupled with a greatly increased excretion of lactate and adipate was compatible with Reye's syndrome. Ketone bodies were not detectable. This case study shows that Reye's syndrome causes altered valproate metabolism, consistent with the defective mitochondrial beta-oxidation of medium chain fatty acids, and suggests that valproic acid should not be used in the treatment of seizures in patients with this syndrome.

Studies of urinary organic acid profiles of a patient with dihydrolipoyl dehydrogenase deficiency.

Using gas chromatography-mass spectrometry (GC/MS), urinary organic acid profile studies were carried out on a patient with dihydrolipoyl dehydrogenase (E3) deficiency. Elevated levels of 2-hydroxyglutaric acid, 2-hydroxyisocaproic acid and 2-oxoisocaproic acid were observed in addition to lactic acid, 2-oxoglutaric acid, 2-hydroxyisovaleric acid and 2-hydroxybutyric acid previously described in patients with E3 deficiency. The 2-oxoglutaric acid levels were significantly lowered after branched-chain amino acid restriction. In an acute period, the patient was slightly ketoacidotic and excreted larger amounts of 2-oxoglutaric acid and lactic acid than in a static period. It was shown that, prior to confirmatory enzyme studies, patients with E3 deficiency who were suspected to have atypical maple syrup urine disease or chronic lactic acidosis can be rapidly identified by GC/MS analysis of urinary acids.

Increased urinary excretion of beta-hydroxyisovaleric acid in ketotic and non-ketotic type II diabetes mellitus.

To evaluate the catabolism of leucine in diabetes mellitus, the urinary excretion of beta-hydroxyisovaleric acid, a by-product of leucine catabolism, in 21 nonproteinuric type II diabetic patients with and without ketosis and 21 control subjects was measured using gas chromatography-mass spectrometry. Urinary beta-hydroxyisovaleric acid and serum leucine concentrations were higher in the 9 ketotic diabetic patients than in the 12 nonketotic diabetic patients (p less than 0.005, p less than 0.01, respectively) or in the control subjects (p less than 0.01, p less than 0.01, respectively). The serum leucine concentrations in the nonketotic diabetic patients and control subjects did not differ significantly (p greater than 0.05), but urinary beta-hydroxyisovaleric acid concentrations were significantly greater in the former (p less than 0.01). These data suggest that in type II diabetic patients the catabolism of leucine is accelerated even in the absence of ketosis and that the urinary beta-hydroxyisovaleric acid concentration is a useful marker of short-term metabolic control in these patients.

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography-mass spectrometry and urease pretreatment.

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography-mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC-MS measurement and provides final results within 1.5 h.

Rapid and sensitive screening for and chemical diagnosis of Canavan disease by gas chromatography-mass spectrometry.

The use of a rapid and sensitive assay for N-acetylaspartate (NAA) in urine or eluates from dried urine on filter paper to make a chemical diagnosis of Canavan disease (CD) is described. It involves a simplified urease pretreatment for sample preparation and gas chromatography-mass spectrometry (EI, scanning mode) with or without stable isotope dilution. Significant improvements in the recovery of NAA and the GC-MS data-handling device made the assay without stable isotope dilution sensitive and quantitative enough to diagnose CD: Its coefficient of variation (CV) was below 12%. The CV obtained with stable isotope dilution was below 9%. One patient with CD had an abnormal NAA level that was more than 6 S.D. above the mean of the age-matched controls. This diagnostic procedure is accurate for screening and for the chemical diagnosis of CD, with a good cost:benefit ratio. The urinary NAA levels of the healthy controls decreased significantly with age. This change should be considered in making a chemical diagnosis of this disease.