13C-phenylalanine breath test and serum biopterin in schizophrenia, bipolar disorder and major depressive disorder.

Phenylalanine is required for the synthesis of the neurotransmitters dopamine, noradrenaline, and adrenaline. The rate-limiting step for phenylalanine metabolism is catalyzed by phenylalanine hydroxylase (PAH) and its cofactor tetrahydrobiopterin. We aimed to detect altered phenylalanine metabolism in major psychiatric disorders using the l-[1-13C]phenylalanine breath test (13C-PBT) and serum biopterin levels. We also investigated association of PAH mutations with schizophrenia and phenylalanine metabolism. 13C-phenylalanine (100 mg) was orally administered, and the breath 13CO2/12CO2 ratio was monitored for 120 min in four groups: 103 patients with schizophrenia (DSM-IV), 39 with bipolar disorder, 116 with major depressive disorder (MDD), and 241 healthy controls. Serum biopterin levels were measured by high performance liquid chromatography. Mutation screening of PAH exons was performed by direct sequencing in 46 schizophrenia patients. Association analysis was performed using six tag single nucleotide polymorphisms and the PAH Arg53His mutation by TaqMan assays in 616 schizophrenia patients and 1194 healthy controls. Analyses of covariance controlling for age, sex, and body weight showed that the index for the amount of exhaled 13CO2 was significantly lower in the schizophrenia group than in the other three groups (all p < 0.05). Biopterin levels in schizophrenia and MDD were significantly lower than those in controls. Biopterin levels correlated with 13C-PBT indices in controls. PAH polymorphisms were not associated with schizophrenia or 13C-PBT indices. 13C-PBT revealed reduced phenylalanine metabolism in schizophrenia, though we obtained no evidence of involvement of PAH polymorphism. Serum biopterin levels were lower in schizophrenia and MDD, warranting further investigation.

(13)C-tryptophan breath test detects increased catabolic turnover of tryptophan along the kynurenine pathway in patients with major depressive disorder.

Altered tryptophan-kynurenine (KYN) metabolism has been implicated in major depressive disorder (MDD). The L-[1-(13)C]tryptophan breath test ((13)C-TBT) is a noninvasive, stable-isotope tracer method in which exhaled (13)CO2 is attributable to tryptophan catabolism via the KYN pathway. We included 18 patients with MDD (DSM-IV) and 24 age- and sex-matched controls. (13)C-tryptophan (150 mg) was orally administered and the (13)CO2/(12)CO2 ratio in the breath was monitored for 180 min. The cumulative recovery rate during the 180-min test (CRR0-180; %), area under the Δ(13)CO2-time curve (AUC; %*min), and the maximal Δ(13)CO2 (Cmax; %) were significantly higher in patients with MDD than in the controls (p = 0.004, p = 0.008, and p = 0.002, respectively). Plasma tryptophan concentrations correlated negatively with Cmax in both the patients and controls (p = 0.020 and p = 0.034, respectively). Our results suggest that the (13)C-TBT could be a novel biomarker for detecting a subgroup of MDD with increased tryptophan-KYN metabolism.

A simple convenient synthesis of L-[4-13C]glutamine.

L-[4-(13)C]Glutamine was synthesized from sodium [2-(13)C]acetate in 12 steps and 18% overall yield. A Wittig reaction of (R)-benzyl 4-formyl-2,2-dimethyloxazolidine-3-carboxylate and ethyl 2-(triphenylphosphoranylidene)[2-(13)C]acetate prepared from D-serine and sodium [2-(13)C]acetate, respectively, gave (4S)-4-(2-ethoxycarbonyl[2-(13)C]vinyl)-2,2-dimethyloxazolidine-3-carboxylic acid α,ß-isopropylidene group, oxidation of the resulting hydroxyl group to a carboxyl group and transamidation of the ester moiety gave L-N-Cbz-[4-(13)C]glutamine (Cbz = benzyloxycarbonyl). Finally, removal of the Cbz group gave L-[4-(13)C]glutamine. L-[4-(13)C]Glutamine can be prepared in fewer steps and higher yield by this method compared with previously reported methods.

The effect of methanol extracts of tsao-ko (Amomum tsao-ko Crevost et Lemaire) on digestive enzyme and antioxidant activity in vitro, and plasma lipids and glucose and liver lipids in mice.

Our previous study showed that tsao-ko intake can lower plasma and liver triacylglycerol (TG) concentrations and has hypoglycemic and antioxidant activity in mice. This study involved separating two major fractions (A and B) from the methanol extracts (MeX) of tsao-ko using silica gel column chromatography, and then determining the effect of the fractions in vivo and in vitro to clarify the most effective components of tsao-ko. An intake of MeX and A fraction statistically significantly reduced body lipids and plasma thiobarbitutic acid reactive substances (TBARS) concentrations compared with the control and inhibited lipase and alpha-glucosidase activities. These reductions were not observed in mice fed the B fraction and these inhibitions of B fraction were mild compared with MeX and A fraction. The plasma and liver TG concentrations of each fraction group did not show significant differences compared with the control. The [M-H](+) and maximum UV absorption of the A fraction were 291 m/z and 279 nm, respectively. The peak of A fraction appeared at a similar time to the epicatechin standard in the LC/MS/MS analysis and the MS/MS spectrum of the A fraction was similar to that of the epicatechin standard. It was concluded that the most effective component of tsao-ko for body lipid reduction and hypoglycemic and antioxidant activity was contained in the polar fraction and the evidence suggested that this component could be epicatechin. However, the strongest TG lowering components of tsao-ko may be methanol insoluble.

Effect of lipid extracted from tsao-ko (Amomum tsao-ko Crevost et Lemaire) on digestive enzyme activity, antioxidant activity, plasma and liver lipids, and blood glucose levels of mice.

Lipids extracted from tsao-ko were separated into three fractions with silica gel column chromatography and fed to mice (3 mo old) for 90 d to clarify their inhibitory activity on digestive enzyme activity. The diets contained the following: control--no tsao-ko, 0.05% total lipid of tsao-ko (TL), 0.0109% chloroform fraction (CF), 0.0245% acetone fraction (AF), or 0.00365% methanol fraction (MeF). Although CF and AF slightly inhibited the activities of alpha-glucosidase, alpha-amylase, and lipase, intakes of these fractions had little influence on plasma and liver lipid concentrations when compared with the control diet. MeF did not inhibit alpha-glucosidase but had DPPH radical scavenging activity and the mice fed this fraction had the most marked reduction in plasma glucose and TBARS concentrations compared with the other diet groups. These results suggest that the fat-soluble polar components of tsao-ko contain an active component that might be associated with decreased plasma glucose and TBARS concentrations in mice.

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ-AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)(1).

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ-aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the (13) C-enrichment ratios ((13) C-ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l-[3-(13) C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%-67%, compared with that in our previous d-[1-(13) C]glucose feeding experiment (Iida et al. 2002). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C-13(2) of chl a was labeled with the (13) C-carbon of l-[methyl-(13) C]methionine derived from l-[3-(13) C]alanine via [2-(13) C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l-[3-(13) C]serine. The phytyl moiety of chl a was also labeled on C-P2, C-P3(1) , C-P4, C-P6, C-P7(1) , C-P8, C-P10, C-P11(1) , C-P12, C-P14, C-P15(1) , and C-P16 from (13) C-isoprene (2-[1,2-methyl,3-(13) C3 ]methyl-1,3-butadiene) generated from l-[3-(13) C]alanine via [2-(13) C]acetyl CoA.

(13)C-phenylalanine breath test correlates with liver fibrosis in postoperative biliary atresia.

BACKGROUND: Values derived from the (13)C-phenylalanine breath test (PBT) may serve as an index for liver fibrosis and clinically predictive readings for liver diseases in adults. In the present study the PBT was conducted in postoperative biliary atresia (BA) children to evaluate phenylalanine metabolism in the liver, and the results based on biochemical data, especially the index on liver fibrosis, were compared with PBT findings. METHODS: Hepatofunctional evaluations were conducted in 10 postoperative BA children with moderate (group B; n = 4) and severe (group A; n = 6) liver dysfunction, and the PBT results were compared with those of 13 normal healthy children (group C). Subjects were orally given single-bolus (13)C-phenylalanine at 3.5 mg/kg (maximum dosing: 100 mg) in the morning. Time-related exhaled gas was periodically collected until 120 min after dosing. The (13)CO(2) levels were monitored with gas chromatography-mass spectrometry before and after administration, and the (13)C excretion rate, (13)C cumulative excretion and time of maximum (13)C excretion rate were monitored accordingly. RESULTS: Total bile acid, hyaluronic acid, type IV collagen 7S, total bilirubin or albumin and the PBT findings were significantly correlated. The PBT findings in group A were significantly lower those of group B, indicating that phenylalanine metabolism was markedly attenuated in the former. CONCLUSION: The PBT values correlated well with liver fibrosis in postoperative BA children. Because PBT is a non-invasive approach, results from this method may serve as a useful and reliable index for post-surgical monitoring of children operated on for liver fibrosis.

Metabolic pathways leading from amino acids to vitamin B12 in Propionibacterium shermanii, and the sources of the seven methyl carbons.

The metabolic pathways leading from l-[2-13C]aspartic acid, [2-13C]glycine and l-[methyl-13C]methionine to vitamin B12 were investigated, focusing on the biosynthetic pathways leading to the aminopropanol moiety of vitamin B12 and on the role of the Shemin pathway leading to delta-aminolevulinic acid (a biosynthetic intermediate of tetrapyrrole), by means of feeding experiments with Propionibacterium shermanii in combination with 13C-NMR spectroscopy. The 13C-methylene carbons of l-[2-(13)C]aspartic acid, which is transformed to [2-13C]glycine via l-[2-13C]threonine, and [2-13C]glycine added to the culture medium served mainly to enrich the seven methyl carbons of the corrin ring through C-methylation by S-adenosyl-l-[methyl-13C]methionine derived from catabolically generated l-[methyl-13C]methionine in the presence of tetrahydrofolic acid. The results indicate that the catabolism of these amino acids predominates over pathways leading to (2R)-1-amino-2-propanol or delta-aminolevulinic acid in P. shermanii. Feeding of l-[methyl-13C]methionine efficiently enriched all seven methyl carbons. In the cases of [2-13C]glycine and l-[methyl-13C]methionine, the 13C-enrichment ratio of the methyl carbon at C-25 (the site of the first C-methylation) was less than those of the other six methyl carbons, probably due to the influence of endogenous d-glucose in P. shermanii. The almost identical 13C-enrichment ratios of the other six methyl carbons indicated that these C-methylations during vitamin B12 biosynthesis were completed before the amino acids were completely consumed. However, in the case of l-[2-13C]aspartic acid, the 13C-enrichment ratios of five methyl carbons were low and similar, whereas the last two sites of C-methylation (C-53 and C-35) were not labeled, presumably because of complete consumption of the smaller amount of added label. The ratios of 13C-incorporation into the seven methyl carbons are influenced by the conditions of amino acid feeding experiments in a manner that is dependent upon the order of C-methylation in the corrin ring of vitamin B12.

Effects of tetrahydrobiopterin and phenylalanine on in vivo human phenylalanine hydroxylase by phenylalanine breath test.

BH(4) administration results in the reduction of blood phenylalanine level in patients with tetrahydrobiopterin (BH(4))-responsive phenylalanine hydroxylase (PAH) deficiency. The mechanism underlying BH(4) response remains unknown. Here, we studied the effects of BH(4) and phenylalanine on in vivo PAH activity of normal controls using the phenylalanine breath test (PBT) by converting l-[1-(13)C] phenylalanine to (13)CO(2). Phenylalanine oxidation rates were expressed as Delta(13)C ((13)CO(2)/(12+13)CO(2), per thousand) and cumulative recovery rates over 120min (CRR(120), %; total amount of (13)CO(2)/the administered dose of (13)C-phenylalanine). Under physiological conditions of blood phenylalanine, BH(4) administration reduced the Delta(13)C peak from 40.8 per thousand to 21.6 per thousand and CRR(120) from 16.9% to 10.2%. Under high blood phenylalanine conditions, administration of BH(4) increased the Delta(13)C peak from 30.7 per thousand to 46.0 per thousand, while the CRR(120) was similar between phenylalanine (19.9%) and phenylalanine+BH(4) (21.1%) groups. Corrected Delta(13)C and CRR(120) were calculated against serum phenylalanine levels to remove the effects of phenylalanine loading. After BH(4) administration, the corrected Delta(13)C peak increased from 82.7 per thousand to 112.6 per thousand, while the corrected CRR(120) was similar (47.6% and 45.6%). These results indicate that phenylalanine worked as a regulator of in vivo PAH by serving as both a substrate and an activator for the enzyme. Excessive dosages of BH(4) inhibited PAH under normal phenylalanine conditions and activated PAH under conditions of high phenylalanine. The regulation system is therefore designed to maintain phenylalanine levels in the human body. Appropriate BH(4) supplementation must be reviewed in patients with BH(4)-responsive PAH deficiency.

Identification of tetrapyrrole compounds excreted by Rhodobacter sphaeroides and sources of the methyl hydrogens of bacteriochlorophyll a biosynthesized by R. sphaeroides, based on 13C-NMR spectral analysis of coproporphyrin III tetramethyl ester.

Red-fluorescent tetrapyrrole compounds excreted by Rhodobacter sphaeroides into the culture broth were concluded to be coproporphyrinogen (Copro'gen) III and uroporphyrinogen (Uro'gen) I, based on the (13)C-NMR spectral identification of coproporphyrin (Copro) III tetramethyl ester and uroproporphyrin (Uro) I octamethyl ester. The sources of the methyl hydrogens of bacteriochlorophyll a were established by analysis of the (13)C-NMR spectra of (2)H,(13)C-Copro III tetramethyl ester chemically derived from (2)H,(13)C-Copro'gen III biosynthesized through the feeding of delta-amino[2-(13)C]levulinic acid (ALA) to R. sphaeroides in medium containing 50% (2)H(2)O. We confirmed the previous finding that one of the methyl hydrogens was derived from water in the medium during decarboxylation of four acetyl side chains of Uro'gen III to generate Copro'gen III. It was further shown that the other hydrogen atoms, previously reported to be derived from methylene hydrogens at C-2 of ALA, had been exchanged with hydrogen of water in the medium in the biosynthetic pathways leading from ALA to Copro'gen III.

Mechanism of the ring contraction process in vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii under aerobic conditions.

The mechanism of the ring contraction process during vitamin B(12) biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with delta-amino[1-(13)C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and delta-amino[1-(13)C,1,1,4-(18)O(3)]levulinic acid in combination with (13)C-NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin-3x from formation of the gamma-lactone from the ring A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ring D by cleavage of the carbon-oxygen bond at C1 of precorrin-3x) in the aerobe Pseudomonas denitrificans was not seen in P. shermanii under aerobic conditions, and the mechanism of the ring contraction process in P. shermanii was the same irrespective of the presence or absence of oxygen.

[Improvement of a multi-target screening analysis for drugs using a QTRAP liquid chromatography/tandem mass spectrometry system].

We have investigated the usefulness of an improved multi-target screening method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis on a hybrid triple-quadrupole linear ion trap mass spectrometer for detecting major drugs in toxicological analysis. Fifteen drugs, most frequently detected in intoxicated patients treated at the emergency medicine section in our institute, were mixed together in serum or urine at concentration of 100 ng/ml each, and then extracted by liquid-liquid extraction using 1-chlorobutane, followed by an analysis using LC/MS/MS. By setting the collision energy at the multiple reaction monitoring mode and declustering potential at the enhanced product ion scan mode individually for each drug, clinically satisfactory sensitivity was attained for the detection of drugs in extracted samples. The results of the present study on several serum/urine samples of intoxicated patients indicated that this method is superior to other screening methods in reliability. Taken together, our improved LC/MS/MS analysis is useful for detecting major drugs causing intoxication in the field of emergency medicine.

Proposal for internet-based Digital Dental Chart for personal dental identification in forensics.

A dental chart is very useful as a standard source of evidence in the personal identification of bodies. However, the kind of dental chart available will often vary as a number of types of odontogram have been developed where the visual representation of dental conditions has relied on hand-drawn representation. We propose the Digital Dental Chart (DDC) as a new style of dental chart, especially for open investigations aimed at establishing the identity of unknown bodies. Each DDC is constructed using actual oral digital images and dental data, and is easy to upload onto an Internet website. The DDC is a more useful forensic resource than the standard types of dental chart in current use as it has several advantages, among which are its ability to carry a large volume of information and reproduce dental conditions clearly and in detail on a cost-effective basis.

Ligand-induced structural changes of the CD44 hyaluronan-binding domain revealed by NMR.

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

In vivo studies of phenylalanine hydroxylase by phenylalanine breath test: diagnosis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency.

Tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency is characterized by reduction of blood phenylalanine level after a BH4-loading test. Most cases of BH4-responsive PAH deficiency include mild phenylketonuria (PKU) or mild hyperphenylalaninemia (HPA), but not all patients with mild PKU respond to BH4. We performed the phenylalanine breath test as reliable method to determine the BH4 responsiveness. Phenylalanine breath test quantitatively measures the conversion of L-[1-13C] phenylalanine to 13CO2 and is a noninvasive and rapid test. Twenty Japanese patients with HPA were examined with a dose of 10 mg/kg of 13C-phenylalanine with or without a dose of 10 mg . kg(-1) . d(-1) of BH4 for 3 d. The phenylalanine breath test [cumulative recovery rate (CRR)] could distinguish control subjects (15.4 +/- 1.5%); heterozygotes (10.3 +/- 1.0%); and mild HPA (2.74%), mild PKU (1.13 +/- 0.14%), and classical PKU patients (0.29 +/- 0.14%). The genotypes in mild PKU cases were compound heterozygotes with mild (L52S, R241C, R408Q) and severe mutations, whereas a mild HPA case was homozygote of R241C. CRR correlated inversely with pretreatment phenylalanine levels, indicating the gene dosage effects on PKU. BH4 loading increased CRR from 1.13 +/- 0.14 to 2.95 +/- 1.14% (2.6-fold) in mild PKU and from 2.74 to 7.22% (2.6-fold) in mild HPA. A CRR of 5 to 6% reflected maintenance of appropriate serum phenylalanine level. The phenylalanine breath test is useful for the diagnosis of BH4-responsive PAH deficiency and determination of the optimal dosage of BH4 without increasing blood phenylalanine level.

[Simple quantitation of arsenic by energy dispersive fluorescence X-ray spectrometer using Reinsch's test].

We examined the clinical usefulness of the Reinsch's test for the detection of the small amounts of the heavy metals such as arsenic and mercury using the fluorescence X-ray spectrometry. We tried t o measure various kinds of biological samples, including serum, urine, and gastric contents using this method. 0.4 ml or 1 ml of hydrochloric acid were added to 2 ml of serum or 6 ml urine and gastric content, respectively, and a copper plate (5.0x 0.8 cm) was immersed into this solution. The mixture heated at 90 degrees C by a heating block for 30 minutes. After heating, the copper plate was washed with water and dried. The copper arsenide that stuck to the copper plate due to Reinsch's test dissolved by methanol/ammonia (8:2) solution at 60 degrees C for 15 minutes. A drop gave the solution to a filter paper fluorescence X-ray analysis and completely dried the filter paper, and applied to the fluorescence X-ray spectrometer. As a result, this method showed about 20 times high sensitivity in comparison with the measurement with condition of solution. The minimal detectable limits of rsenic was 0.4 ppm in serum and were 0.2 ppm in rine and gastric content. The calibration curve could be made for 0.5 to 50 ppm. It will take about 90 min for the measurement using this method for the detection of arsenic in biological samples. We showed the usefulness of the Reinsch's test using the fluorescence X-ray spectrometry in the clinical toxicology.

DNA analysis of neonatal human remains wrapped and kept in a vinyl bag for 15 years.

DNA analysis of a newborn baby wrapped and kept in a vinyl bag for 15 years was performed. DNA isolated from the femur and humerus was used to determine the sex and kinship between the infant and the putative parents. Amplification of mtDNA, ABO, HLA, CST3, CST5, VWA, D12S66, D21S11, CSF1PO, TPOX, THO1 and 10Y polymorphisms and the amelogenin gene was carried out. Several mtDNA types were obtained, suggesting that the sample was contaminated by exogenous DNAs. One of the DNA samples obtained from the femur showed an identical mtDNA sequence to that of the mother except for one site, and this pattern was also found in another DNA sample. None of our laboratory personnel had that type, so we thought it was possible that this sample contained the target DNA. However, maternity was denied by the CST3 polymorphism. Finally, we concluded that the sample had been contaminated with exogenous DNA before we started to examine the body. Although it is difficult to determine the sources of this contamination, PCR amplification from highly degraded DNA is very sensitive to such contamination, and we must be even more careful in DNA analysis of such samples than in that of not so severely degraded specimens.

Hyaluronan recognition mode of CD44 revealed by cross-saturation and chemical shift perturbation experiments.

CD44 is the main cell surface receptor for hyaluronic acid (HA) and contains a functional HA-binding domain (HABD) composed of a Link module with N- and C-terminal extensions. The contact residues of human CD44 HABD for HA have been determined by cross-saturation experiments and mapped on the topology of CD44 HABD, which we elucidated by NMR. The contact residues are distributed in both the consensus fold for the Link module superfamily and the additional structural elements consisting of the flanking regions. Interestingly, the contact residues exhibit small changes in chemical shift upon HA binding. In contrast, the residues with large chemical shift changes are localized in the C-terminal extension and the first alpha-helix and are generally inconsistent with the contact residues. These results suggest that, upon ligand binding, the C-terminal extension and the first alpha-helix undergo significant conformational changes, which may account for the broad ligand specificity of CD44 HABD.

Solid-supported Robinson annulation under microwave irradiation.

Robinson annulation on alumina occurred efficiently on heating with microwave irradiation.