4-hydroxyphenyllactic Acid Research Articles

Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases.

Sweet basil (Ocimum basilicum) peltate glandular trichomes produce a variety of small molecular weight phenylpropanoids, such as eugenol, caffeic acid, and rosmarinic acid, that result from meta hydroxylation reactions. Some basil lines do not synthesize eugenol but instead synthesize chavicol, a phenylpropanoid that does not contain a meta hydroxyl group. Two distinct acyltransferases, p-coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase and p-coumaroyl-coenzyme A:4-hydroxyphenyllactic acid p-coumaroyl transferase, responsible for the production of p-coumaroyl shikimate and of p-coumaroyl 4-hydroxyphenyllactate, respectively, were partially purified and shown to be specific for their substrates. p-Coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase is expressed in basil peltate glands that are actively producing eugenol and is not active in glands of noneugenol-producing basil plants, suggesting that the levels of this activity determine the levels of synthesis of some meta-hydroxylated phenylpropanoids in these glands such as eugenol. Two basil cDNAs encoding isozymes of cytochrome P450 CYP98A13, which meta hydroxylates p-coumaroyl shikimate, were isolated and found to be highly similar (90% identity) to the Arabidopsis homolog, CYP98A3. Like the Arabidopsis enzyme, the basil enzymes were found to be very specific for p-coumaroyl shikimate. Finally, additional hydroxylase activities were identified in basil peltate glands that convert p-coumaroyl 4-hydroxyphenyllactic acid to its caffeoyl derivative and p-coumaric acid to caffeic acid.

CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4′-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4′-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.

An Elicitor-Inducible NADP-Malic Enzyme in Lithospermum erythrorhizon Cultured Cells: cDNA Cloning and Characterization.

The differential display technique was used to isolate genes expressed during yeast extract-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures, and a cDNA clone whose deduced amino acid sequence is similar to plant cytosolic NADP-malic enzymes was identified and designated LeME. The heterologously-expressed LeME oxidized L-malate in presence of NADP, but it did not catalyze formation of 4-hydroxyphenyllactic acid from 4-hydroxyphenylpyruvate in presence of NADPH, one of the crucial biosynthetic steps leading to rosmarinic acid. The potential role of LeME in elicitor-inducible rosmarinic acid biosynthesis is discussed.

Lactic Acid Dimer: An Artifact in the Gas Chromatographic Analysis of Urine with Massive Lactic Acid Aciduria

In severe lactic acidosis (1)(2)(3)(4), secondary increases of pyruvic acid, 2-hydroxybutyric acid, and 4-hydroxyphenyllactic acid are common (5). We describe such a case in which we also found lactic acid dimer. A premature baby, born at 31 weeks of gestation, had hyperbilirubinemia, sepsis, anemia, thrombocytopenia, dermatitis, and metabolic acidosis. Gas chromatography–mass spectrometry (GC-MS) of urine (6) from the infant revealed the following concentrations: lactic acid, 83 100 …

Chromatographic determination of the absolute configuration of an acyclic secondary alcohol using difluorodinitrobenzene

A chromatographic determination method was developed for the absolute configuration of an acyclic secondary alcohol using the characteristic functions of 1,5-difluoro-2,4-dinitrobenzene (FFDNB). This method relies on the formation of the fixed favorable conformation of a secondary alcohol-DLA (2,4-dinitrophenyl-5-leucinamide) derivative and its recognition by ODS silica gel. The secondary alcohol reacted first with FFDNB under mild basic conditions, and L-leucinamide or DL-leucinamide was then introduced into the secondary alcohol-FDNB derivative. Because the conformations of the resulting alcohol-DLA derivatives were rigidly fixed by the dinitrobenzene plane, the absolute configuration at the asymmetric carbon of the secondary alcohol tested can be definitely deduced by the elution behavior of both of the diastereomers in the HPLC and/or LC/MS. One of the diastereomers has the cis (Z)-type arrangement of two more hydrophobic substituents of the alcohol and leucinamide moieties, the more hydrophobic side chain and isobutyl groups, to the plane of the dinitrobenzene, whereas another diastereomer has the opposite arrangement (trans (E)-type). Therefore, the cis arrangement interacts more strongly with the ODS silica gel and has a longer retention time than that of the trans-type arrangement. This established method was successfully applied to various chiral acyclic secondary alcohols including chloramphenicol and 4-hydroxyphenyllactic acid. Finally, the limitations of this method were also examined.

Urine screening of five-day-old newborns: metabolic profiling of neonatal galactosuria

We determined urinary galactose and 4-hydroxyphenyllactic acid (4HPLA) in 4338 of 5-day-old newborns using a newly developed GC–MS screening method. Fifty-two infants were chemically diagnosed as having transient galactosuria based upon elevated urinary galactose levels (4.78–30.53 mg/mg creatinine, control 1.10±0.89 mg/mg creatinine). These infants did not excrete galactitol or galactonic acid into the urine, which is typical of hereditary galactosemia. Nearly 40% of the transient galactosuria was associated with immature infants (low birth weight or borne before 37 gestational weeks). Immature hepatic function is one explanation for neonatal transient galactosuria, but heterozygotes or the carriers of galactose degradation enzyme deficiencies were also suspected in some of the newborns, judging from the comparisons of urinary galactose and 4HPLA excretion between neonates and patients with galactosemia.

4-Hydroxybenzoic acid: a likely precursor of 2,4,6-tribromophenol in Ulva lactuca

Abstract The green marine alga Ulva lactuca is known to contain simple bromophenols, especially 2,4,6-tribromophenol, but the precursor of these compounds in the alga is not known. With the aim of identifying potential precursors, the alga was analyzed for the presence of phenolic compounds. The compounds identified by gas chromatography–mass spectrometry were phenol, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenyllactic acid, 4-hydroxybenzaldehyde, 3,5-dibromo-4-hydroxybenzoic acid and 2,4,6-tribromophenol. Free l -tyrosine and free l -phenylalanine were also present in the alga. Furthermore, a crude enzyme extract from the alga, which contained bromoperoxidases, was used to brominate a range of phenolic compounds and the formation of bromophenols was monitored. The compounds forming 2,4,6-tribromophenol were phenol, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzyl alcohol and 2-hydroxybenzyl alcohol. 4-Hydroxybenzoic acid is designated as the most likely precursor of 2,4,6-tribromophenol in U. lactuca and a pathway for its formation from l -tyrosine, via 4-hydroxyphenylpyruvic acid, is proposed.

Increased Excretion of Aromatic Amino Acid Catabolites in Animals Infected with Trypanosoma brucei evansi

Aromatic amino acid catabolism by Trypanosoma brucei evansi was investigated in vivo using C3HeB/FeJ mice. The major catabolites detected by gas chromatography in the urines of infected animals were phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, and indole-3-pyruvic acid. Identity of each compound was confirmed by gas chromatography-mass spectrometry. Concentrations of catabolites in urine of infected mice were correlated with parasitemia and returned to normal following suramin treatment. Other aromatic amino acid metabolites, including indole-3-acetic acid, indole-3-lactic acid, and 4-hydroxyphenyllactic acid, were detected in urine from infected animals by gas chromatography mass spectrometry, although quantities were too low to be quantified reproducibly. Both phenylpyruvic acid and 4-hydroxyphenylpyruvic acid were also detected in urine of dogs and donkeys experimentally infected in Egypt with a recent field isolate of T. b. evansi. Tryptophan metabolites could not be assayed in dog and urine samples because formalin, which degraded the indole acids, had to be added before the samples could be imported into the U.S. Finally, concentrations of urinary catabolites during infection were correlated with the tyrosine aminotransferase activity in infected mouse sera.

152 INCREASED URINARY EXCRETION OF DICARBOXYLIC ACIDS AND 4 HYDROXYPHENYLLACTIC ACID IN PATIENTS WITH ZELLWEGER SYNDROME

s-Oxidation of dicarboxylic acids has been shown to occur in mitochondria iind peroxisomes of rat liver in vitro. Patients with Zellweger syndrome (ZS) offer a unique possibility to study the importance of peroxisomes in the degradation of dicarboxylic acids in vivo. Methods: We quantified urinary organic acids in seven ZS patients with impaired peroxisomal s-oxidation but normal mitochondrial s-oxidation capacity by GCMS. Results: Increased amounts of dicarboxylic acids with an odd number of carbon atoms (e.g. azelaic (up to 23 vs <0.1 mmol/mol creatinine in controls), pimelic acid (up to 15 vs <0.1)) or an even number of carbon atoms (e.g. adipic (up to 180 vs <0.1 nmol/mol creatinine in controls), suberic (up to 140 vs <0.1), sebacic acid (up to 200 vs <0.1)) and 2-hydroxysebacic acid, a medium-chain hydroxydicarboxylic acid (up to 50 vs <0.1 mmol/mol creatinine in controls), were found in the urines of these patients. In addition, patients with ZS Slowed an highly elevated excretion of 4-hydroxyphenyllactic acid (up to 98 vs <0.1 mmol/mol creatinine in controls). Conclusion: These results prove the functional importance of peroxisomes in the s-oxidation of these compounds in vivo. The characteristic pattern of urinary organic acids in ZS should prompt for specific diagnostic procedures for peroxisomal disorders, when found in patients undergoing selective screening for inherited metabolic diseases.

A study of urinary metabolites in patients with dicarboxylic aciduria for differential diagnosis

Dicarboxylic aciduria (DCA-uria) is a relatively common finding in the screening of organic acidemias by gas chromatography/mass spectrometry (GC/MS). A considerable number of patients with DCA-uria are involved in disturbances of mitochondrial and peroxisomal fatty acid beta-oxidation. The differential diagnosis of DCA-uria was investigated using a combination of organic acid analysis by GC/MS, carnitine determination, acylcarnitines by fast atom bombardment/mass spectrometry (FAB/MS) and acylglycines by stable-isotope dilution analysis. The relative distribution of urinary metabolites was examined in 46 patients with DCA-uria of different origins, including physiological ketosis of childhood, disorders of propionic acid metabolism, glutaric aciduria type II, Zellweger syndrome and patients who were clinically diagnosed as having Reye syndrome. Zellweger syndrome seemed to be distinguishable from other disorders by the high sebacic acid/adipic acid ratio of DCA-uria and increased excretion of 4-hydroxyphenyllactic acid and 2-hydroxysebacic acid. The mild form of glutaric aciduria type II was often missed by current organic acid analysis alone, but was readily diagnosed by acylcarnitine and acylglycine determination. The ratio of free/total carnitine was low in most of the DCA-uria patients except for two of five cases of Zellweger syndrome and one of three cases of Reye syndrome. The acylcarnitine analysis by FAB/MS showed adipyl-, suberyl-, sebacyl- or dodecanedioylcarnitine as major peaks in most of these patients, although these were not specific. Disease-specific peaks were detectable only in congenital organic acidemias such as glutaric aciduria type II, methylmalonic acidemia and propionic acidemia.

Identification of the reaction products of rosmarinic acid synthase from cell cultures of Coleus blumei by ion spray mass spectrometry and tandem mass spectrometry

AbstractDifferent substrates were used in reaction assays for rosmarinic acid synthase, an enzyme involved in rosmarinic acid biosynthesis in cell cultures of Coleus blumei. In order to introduce the cinnamoyl moiety of the ester, 4‐coumaroyl‐CoA and caffeoyl‐CoA were applied as substrates; for the hydroxyphenyllactic acid moiety, 4‐hydroxyphenyllactate and 3,4‐dihydroxyphenyllactate were used. Four different reaction products were thus produced and these were analysed by ion spray mass spectrometry and tandem mass spectrometry after purification by semi‐preparative high pressure liquid chromatography. The molecular masses and the fragmentation patterns obtained by tandem mass spectrometry of the reaction products formed by the catalysis of rosmarinic acid synthase were in agreement with the structures of rosmarinic acid (caffeoyl‐3′,4′‐dihydroxyphenyllactate) and rosmarinic acid‐like esters (4‐coumaroyl‐4′‐hydroxyphenyllactate, 4‐coumaroyl‐3′,4′‐dihydroxyphenyllactate, caffeoyl‐4′‐hydroxyphenyllactate) as expected for the substrates used in the enzyme reaction.

Determination of creatinine and ultraviolet-absorbing amino acids and organic acids in urine by reversed-phase high-performance liquid chromatography

A simple and reliable method for the determination of urinary creatinine, amino acids and organic acids was developed. A urine sample was preliminarily separated into an organic acid fraction (including neutral species) and an amino acid fraction by cation-exchange chromatography. Both fractions were analysed by reversed-phase high-performance liquid chromatography, with a phosphoric acid—methanol gradient elution system and ultraviolet detection at 210 nm. Relationships between concentrations and peak heights were linear from 2 to 500 μ M for the analytes. Overall recoveries were ca. 100%. The concentrations of creatinine for 37 urine samples, from 20 healthy newborns and from 17 patients with several inherited metabolic disorders, were 2.35 ± 2.29 m M (ranging between 0.27 and 10.15 m M). The method was applied to the determination of several diagnostically useful metabolites in urine. The concentrations of phenylalanine and phenylacetic acid for five urine samples from patients with phenylketonuria were 347 ± 177 and 282 ± 224 μ M/m M creatinine, respectively. The concentrations of tyrosine and 4-hydroxyphenyllactic acid in the urine of a patient with tyrosinemia were 112 and 1871 μ M/m M creatinine, respectively.

Screening urine of 3-week-old newborns: transient methylmalonic and hydroxyphenyllactic aciduria.

Urinary methylmalonic acid (MMA) and 4-hydroxyphenyllactic acid (HPL) have been determined in 3345 and 2498 3-week-old newborns, respectively. Urine was collected onto filter paper and assayed by a rapid gas chromatography-mass spectrometry method. Forty-six infants (1.7%) had elevated MMA levels (greater than 58.5 micrograms/mg creatinine, means + 5 SD) and 31 infants (1.2%) had elevated levels of HPL (greater than 87.7 micrograms/mg creatinine, means + 5 SD). Fifteen infants with elevated values of MMA were retested from one to several months after the first test. In 12 infants the MMA levels normalized, while in the remaining three, elevated methylmalonic acid persisted. Nine infants with elevated values of HPL were retested, and in all except one, HPL levels normalized. No access to clinical evaluation of the infants was available. Transient methylmalonic aciduria and transient tyrosyluria affect a substantial number of infants and the clinical significance of this phenomenon has yet to be determined.

Neurotransmitters in neocortex of aged rhesus monkeys

The effects of aging on levels of neurotransmitters were determined in two regions of the cerebral cortex in rhesus monkeys ( Macaca mulatta). Choline acetyltransferase (ChAT) activity as well as somatostatin, neuropeptide Y, and substance P immunoreactivities were analyzed in the right caudal cingulate gyrus and in the left and right inferior occipital poles in five age groups: 4–6 years; 8–11 years; 20–25 years; 26–29 years; and 31–34 years. Neuroactive amino acids and markers for monoamine transmitters were analyzed only in the youngest (4–6 years) and oldest (31–34 years) animals. Across the five age groups studied, ChAT activity as well as somatostatin and neuropeptide Y immunoreactivities were significantly decreased bilaterally in occipital poles of the 31- to 34-year-old group. There were no significant age-related differences in substance P immunoreactivity. In 4–6-year-old vs. 31–34-year-old monkeys, levels of amino acid neurotransmitters were unchanged. However, there were significant reductions in norepinephrine, serotonin and its metabolites, kynurenine, and 4-hydroxyphenyllactic acid in occipital poles of the 31- to 34-year-old monkeys. No significant neurochemical changes were detected in the cingulate cortex. These findings demonstrate that aged nonhuman primates show reductions in cortical markers for a variety of neurotransmitters, including acetylcholine, somatostatin, neuropeptide Y, norepinephrine, and serotonin but that these changes do not occur uniformly in the neocortex.

Characterization of rosmarinic acid synthase from cell cultures of Coleus blumei

Rosmarinic acid synthase (RAS) catalyses the transesterification reaction of CoA-activated cinnamic acids, e.g. 4-coumaroyl- or caffeoyl-CoA, and hydroxyphenyllactic acids, e.g. 4-hydroxy- or 3,4-dihydroxyphenyllactic acid, during the biosynthesis of rosmarinic acid. The enzyme isolated from suspension cultures of Coleus blumei is soluble and has an optimal pH of 7.0–7.5. Michaelis-Menten kinetics were observed with respect to the substrates 4-coumaroyl- and caffeoyl-CoA with K m -values of 20 and 33 μM, respectively. Only R(+)-stereoisomers of the hydroxyphenyllactic acid are accepted by RAS. For 4-hydroxy- and 3,4-dihydroxyphenyllactic acid the K m -values were 0.17 and 0.37 mM, respectively. The RAS reaction was inhibited by 4-hydroxymercuribenzoate, the S(−)-stereoisomer of 3,4-dihydroxyphenyllactic acid, hydroxyphenylpyruvates and rosmarinic acid, whereas cinnamic acids were not inhibitory. Coenzyme A showed a non-competitive inhibition. The reverse reaction of RAS, the splitting of rosmarinic acid with help of coenzyme A into caffeoyl-CoA and 3,4-dihydroxyphenyllactate, was readily detected. The kinetic data for this reaction are K m -values of 310 μM for coenzyme A and 15 μM for rosmarinic acid.

Urinary organic acid excretion by babies born before 33 weeks of gestation.

Using analytical procedures that are widely used by laboratories investigating metabolic disorders, we investigated urinary organic acid excretion by premature neonates who were receiving the usual clinical care. Our purpose was to provide a basis for the diagnosis of inherited organic acid defects. We analyzed 127 random (untimed) urine samples collected weekly from 22 infants of 25-32 weeks of gestation (median, 28 weeks). A wide variety of organic acids was excreted. After oximation, they were extracted with ethyl acetate and diethyl ether, derivatized to trimethylsilyl forms, and analyzed by gas-liquid chromatography on a nonpolar fused silica capillary column, with mass spectrometry for identification. Profiles for individual babies varied markedly on different occasions, reflecting their metabolic status and bacterial activity in the gut. There was no significant ketonuria. Three metabolites identified for the first time in urine from normal neonates were 2,3-butanediol, 3-hydroxy-2-butanone (acetoin), and 4-hydroxy-3-methoxyphenyllactic acid. Significantly increased excretion of 4-hydroxyphenyllactic acid and other phenolic acids occurred during parenteral feeding.

Gas chromatographic profiling of phenolic acids in urine of patients with chirrhosis of the liver

Phenolic acids are analysed within the profile of the organic acids in urine of patients with cirrhosis. For the following constituents an increased urinary excretion is observed: 4- hydroxyphenylpyruvic acid, 4-hydroxyphenyllactic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 4-hydroxyhippuric acid, vanillic acid, homovanillic acid, 4-hydroxymandelic acid, 4-hydroxy-3-methoxyphenylpropionic acid and p-cresol. The phenols are metabolites of tyrosine and are produced in the liver, in extrahepatic tissues and by intestinal microorganisms. They are suggested as biochemical control parameters for the metabolizing function of the liver, for the effect of therapy and for the existence of portal-systemic venous collaterals.

Chemical analysis of succinylacetone and 4-hydroxyphenyllactate in amniotic fluid using selective ion monitoring.

A method for the measurement of the concentration of succinylacetone and 4-hydroxyphenyllactic acid in amniotic fluid was developed for the prenatal diagnosis of hereditary tyrosinemia. Succinylacetone was converted to 5-methyl-3-isoxazolepropionic acid and isolated with 4-hydroxyphenyllactic acid by liquid partition chromatography and the trimethylsilyl derivatives quantified by ammonia chemical ionization selected ion monitoring gas chromatography-mass spectrometry with 2-hydroxy-n-caproic acid as the internal standard. The concentration of 4-hydroxyphenyllactic acid in normal amniotic fluid was 1.97 +/- 0.75 (S.D.) mumol/l while succinylacetone was undetectable. A pregnancy at risk for tyrosinemia type II was monitored. The concentration of 4-hydroxyphenyllactic acid was within the normal range and a healthy child was born.

Interactions between parallel pathways during biosynthesis of rosmarinic acid in cell suspension cultures of Coleus blumei

Phenylalanine ammonia-lyase from an "over-producer" strain of Coleus blumei Benth. cell cultures accumulating high levels of rosmarinic acid (RA) has been shown to possess no special feed-back sensitivity to RA or its precursors. No tyrosine-3-hydroxylase activity could be detected in culture extracts and no specific inhibitors of tyrosine incorporation into RA were found. L-α-aminooxy-β-phenyl propionic acid, however, was effective in specifically blocking phenylalanine incorporation. This block also led to an accumulation of label from tyrosine in 4-hydroxyphenyllactic acid rather than in 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenyllactic acid. These observations require a re-evaluation of the possible role of DOPA as a major biogenic precursor to RA.

Study of the intestinal tyrosine metabolism using stable isotopes and gas chromatography-mass spectrometry

Deuterated tyrosine, 4-hydroxyphenyllactic acid, 4-hydroxyphenylpropionic acid, 4-hydroxyphenylacetic acid and 4-hydroxybenzoic acid were incubated under anaerobic conditions with human faecal specimens for the in vitro study of their respective metabolisms. After 1 week, aromatic acids and phenols were extracted and analyzed by gas chromatography-mass spectrometry. [3′,5′- 2H 2]Tyrosine produced 4-hydroxyphenyllactic acid, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid; [3′,5′- 2H 2]-4′-hydroxyphenyllactic acid produced 4-hydroxyphenylpropionic acid, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylacetic acid and phenylpropionic acid; [3′,5′- 2H 2]-4′-hydroxyphenylpropionic acid produced 3-hydroxyphenylpropionic acid and phenylpropionic acid; [3′,5′,2,2- 2H 4]-4′-hydroxyphenylacetic acid produced p-cresol; and [3′,5′- 2H 2]-4′-hydroxybenzoic acid produced phenol. Thus the intestinal flora showed activities for decarboxylation leading to phenol and p-cresol, dehydroxylation leading to phenylpropionic acid and rearrangement leading to 3-hydroxyphenylpropionic acid. Retention of both deuterium labels was observed in the rearrangement reaction.