Metabolic Studies and Clinical Observations During L-Dopa Treatment of Parkinson's Disease

Twenty-two patients with Parkinson''s disease were treated for the periods of up to six months with L-dopa. In nine of the male patients metabolic observations were made after oral administration of ¹⁴C-L-dopa.Peak serum levels of total radioactivity represented small fractions of the dose given and occurred at one to two hours after ingestion. Two-thirds of the dose was excreted as metabolites in urine in eight hours. Insignificant fractions of the dose were excreted in stool and expired air. These results indicate rapid and complete absorption from the gastrointestinal tract, as well as rapid distribution and excretion. Clinical observations confirmed that L-dopa is an effective treatment for Parkinson''s disease. Improvements in disability averaged 47% at 30 days, 55% at 50 days, and 60% at three months. Degree of improvement tended to be inversely related to age of patient, duration of illness, and severity of disease. Side-effects were seen in most patients, but were always reversible with dose reduction. Nausea was the chief dose-limiting side-effects in early therapy and choreoathetosis after two months of treatment. The average tolerated daily dose was 3 g. On the basis of this experience it seems that the drug can be used safely and effectively on an outpatient basis provided that dosage increments are introduced gradually, maximum dosage is limited to 4 g. a day, and supervision is both close and continuous.     

L-Dopa Therapy in Parkinson's Disease: A Critical Review of Nine Years' Experience

The last 10 years have seen great activity in the investigation of cerebral catecholamines, particular attention having been paid to dopamine. The low dopamine content in the basal ganglia and in the urine of patients with Parkinson''s disease led to the logical use of the precursor DOPA in the treatment of this disorder. Between 1961 and 1966, both the oral and the intravenous routes were utilized and some effects were noted upon akinesia and rigidity. The doses then used were low and the results remained somewhat controversial. When higher oral levels of L-dopa were introduced, the beneficial action of L-dopa upon parkinsonian symptoms and signs was proved beyond doubt, but there came to light a number of troublesome side effects, the worst of which were hypotension and a variety of abnormal involuntary movements. Recently, new approaches to the therapy have been tried and the sum total of these observations is to challenge our peace of mind regarding a seemingly logical chain of events. We are convinced that such second thoughts will eventually result in better and safer methods of treating this too frequent and disabling neurological disorder.     

Semi-industrial scale (30 m<Superscript>3</Superscript>) fed-batch fermentation for the production of <Emphasis Type="SmallCaps">d</Emphasis>-lactate by <Emphasis Type="Italic">Escherichia coli</Emphasis> strain HBUT-D15

d(?)-lactic acid is needed for manufacturing of stereo-complex poly-lactic acid polymer. Large scale d-lactic acid fermentation, however, has yet to be demonstrated. A genetically engineered Escherichia coli strain, HBUT-D, was adaptively evolved in a 15% calcium lactate medium for improved lactate tolerance. The resulting strain, HBUT-D15, was tested at a lab scale (7 L) by fed-batch fermentation with up to 200 g L^?1 of glucose, producing 184–191 g L^?1 of d-lactic acid, with a volumetric productivity of 4.38 g L^?1 h^?1, a yield of 92%, and an optical purity of 99.9%. The HBUT-D15 was then evaluated at a semi-industrial scale (30 m³) via fed-batch fermentation with up to 160 g L^?1 of glucose, producing 146–150 g L^?1 of d-lactic acid, with a volumetric productivity of 3.95–4.29 g L^?1 h^?1, a yield of 91–94%, and an optical purity of 99.8%. These results are comparable to that of current industrial scale l(+)-lactic acid fermentation.     

Treatment of Parkinson's Disease with L-Dopa: A Current Appraisal

Interest in l-dopa therapy for Parkinson''s disease has been considerably enhanced since the recent release of this drug to all medical practitioners. Our experience in the use of l-dopa in 83 patients who have been treated during the past 22 months is presented to provide a practical approach to the administration of l-dopa. Many parkinsonian patients can be treated advantageously on an outpatient basis without the need for initial hospitalization. Some of the common side effects of l-dopa administration can be averted or controlled by a cautious and slow build-up to the optimal dosage level. In the majority (78%) of parkinsonian patients who had been carefully selected for treatment the drug had a beneficial effect on akinesia and rigidity. In the remainder, therapy had to be discontinued because of undesirable side effects or a limited response.     

Quinone and oxyradical scavenging properties of N-acetylcysteine prevent dopamine mediated inhibition of Na+, K+-ATPase and mitochondrial electron transport chain activity in rat brain: Implications in the neuroprotective therapy of Parkinson's disease

Dopamine oxidation products such as H₂O₂ and reactive quinones have been held responsible for various toxic actions of dopamine, which have implications in the aetiopathogenesis of Parkinson's disease. This study has shown that N-acetylcysteine (0.25–1 mm) is a potent scavenger of both H₂O₂ and toxic quinones derived from dopamine and it further prevents dopamine mediated inhibition of Na⁺,K⁺-ATPase activity and mitochondrial respiratory chain function. The quinone scavenging ability of N-acetylcysteine is presumably related to its protective effect against dopamine mediated inhibition of mitochondrial respiratory chain activity. However, both H₂O₂ scavenging and quinone scavenging properties of N-acetylcysteine probably account for its protective effect against Na⁺,K⁺-ATPase inhibition induced by dopamine. The results have important implications in the neuroprotective therapy of sporadic Parkinson's disease since inactivation of mitochondrial respiratory activity and Na⁺,K⁺-ATPase may trigger intracellular damage pathways leading to the death of nigral dopaminergic neurons.     

Alterations in Tyrosine Hydroxylase and Monoamine Oxidase Activity in Blood Vessels

THE enzyme tyrosine hydroxylase¹ (TH), which has been reported as the rate limiting step in noradrenaline biosynthesis, can be modified by nerve stimulation, cold^2,3, exercise⁴, reser-pine, phenoxybenzamine and monoamine oxidase inhibitors^5–7. These treatments affect not only the enzyme in vitro but also catecholamine synthesis in vivo. Much of this information has come from studies with heart, brain, adrenals and spleen, but we found that blood vessels contain appreciable concentrations of noradrenaline⁸ and synthesize it in vivo from its precursor tyrosine. We now report that blood vessels have higher tyrosine hydroxylase activity than the heart and that this activity can be modified by reserpine and L-dihydroxyphenylalanine (L-dopa). Furthermore, the activity of tyrosine hydroxylase in the blood vessels of a spontaneously hypertensive rat differs from that in its normotensive control. We also found that the activity of the enzyme monoamine oxidase in the vasculature was affected by drugs and changes in blood pressure.     

NOREPINEPHRINE METABOLISM IN THE CENTRAL NERVOUS SYSTEM OF MAN: STUDIES USING 3-METHOXY-4-HYDROXYPHENYLETHYLENE GLYCOL LEVELS IN CEREBROSPINAL FLUID

—Levels of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), a major metabolite of norepinephrine, were measured in human CSF by gas-liquid chromatography. MHPG concentrations were similar in both ventricular and lumbar CSF samples; about 30 per cent of the MHPG from either source occurred as the sulphate conjugate. There was relatively little entry of intravenously infused [¹⁴C]MHPG into lumbar spinal fluid. Both α-methylparatyrosine, an inhibitor of tyrosine hydroxylase, and fusaric acid, an inhibitor of dopamine-β-hydroxylase, significantly diminished MHPG values. On the other hand, doses of l -DOPA or probenecid, sufficient to substantially elevate CSF levels of the dopamine metabolite, homovanillic acid, failed to alter the spinal fluid content of MHPG. CSF concentrations of MHPG in patients with Parkinson's disease or the other central nervous system disorders studied did not differ significantly from control levels. The results suggest that MHPG values in CSF may provide an index to norepinephrine metabolism in the central nervous system of man.     

Engineering rTCA pathway and C4-dicarboxylate transporter for <Emphasis Type="SmallCaps">l</Emphasis>-malic acid production

l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L^?1, 0.30 g g^?1, and 0.32 g L^?1 h^?1 in the resulting strain W4209 with CaCO₃ as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L^?1, 0.31 g g^?1, and 0.32 g L^?1 h^?1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.     

Amine Metabolites in the Lumbar Cerebrospinal Fluid of Humans with Restricted Flow of Cerebrospinal Fluid

DETERMINATION of homovanillic acid (HVA) and 5-hydroxy-indole acetic acid (5HIAA) in human lumbar cerebrospinal fluid (CSF) is becoming an important tool in the study of the metabolism in the brain of their respective precursors, dopamine and 5-hydroxytryptamine and in the interpretation of the effects of drugs on these substances. The assumption that the concentration of the acidic metabolites HVA and 5HIAA in the lumbar CSF gives a measure of the amount of turnover of the parent amines in the brain is supported by several findings. (1) Amine metabolite concentrations in the lateral ventricular CSF of the dog correlate with their concentrations in adjacent brain areas¹. (2) Peripherally administered HVA only penetrates slightly or not at all to lateral ventricular CSF in the cat² or dog³, similar results being obtained for 5HIAA in the dog⁴. (3) Drugs which alter brain amine turnover in laboratory animals also alter the concentrations of the acidic metabolites in dog³, rabbit⁵ and human⁶ CSF in the appropriate direction. (4) In Parkinsonism and in senile and presenile dementia, conditions in which there is evidence of defective turnover of amines in the brain, low concentrations of HVA and 5HIAA are found in the CSF⁷.     

Conjugated 3,4 dihydroxy phenyl acetic acid (DOPAC) in human and monkey cerebrospinal fluid and rat brain and the effects of probenecid treatment

Gas chromatography-mass spectrometry (GC-MS) was used to measure 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in cerebrospinal fluid from humans and monkeys and in rat caudate nuclei. DOPAC was found to be present mainly in conjugated form. In human lumbar CSF the average concentration of total DOPAC before probenecid treatment was 1.48 ± 0.31 ng/ml; after probenecid it increased to 15.06 ± 3.17 ng/ml. This increase was mainly due to conjugated DOPAC but increases in free DOPAC also occurred. There is a relatively greater accumulation of DOPAC than of HVA, suggesting that in human CSF conjugated DOPAC may have a faster turnover rate than HVA. In monkey, ventricular CSF contained higher concentrations of DOPAC and HVA than did lumbar CSF.In rat brain, treatment with probenecid caused increases in DOPAC, HVA and their conjugates.These results suggest that DOPAC is conjugated in brain and that both compounds are removed from brain and CSF by a probenecid-sensitive acid transport system in the same manner as is HVA.     

Sweet Sorghum Juice and Bagasse as Feedstocks for the Production of Optically Pure Lactic Acid by Native and Engineered <Emphasis Type="Italic">Bacillus coagulans</Emphasis> Strains

Sweet sorghum is a bioenergy crop that produces large amounts of soluble sugars in its stems (3–7 Mg ha^?1) and generates significant amounts of bagasse (15–20 Mg ha^?1) as a lignocellulosic feedstock. These sugars can be fermented not only to biofuels but also to bio-based chemicals. The market potential of the latter may be higher given the current prices of petroleum and natural gas. The yield and rate of production of optically pure d-(?)- and l-(+)-lactic acid as precursors for the biodegradable plastic polylactide was optimized for two thermotolerant Bacillus coagulans strains. Strain 36D1 fermented the sugars in unsterilized sweet sorghum juice at 50 °C to l-(+)-lactic acid (～150 g L^?1; productivity, 7.2 g L^?1 h^?1). B. coagulans strain QZ19-2 was used to ferment sorghum juice to d-(?)-lactic acid (～125 g L^?1; productivity, 5 g L^?1 h^?1). Carbohydrates in the sorghum bagasse were also fermented after pretreatment with 0.5 % phosphoric acid at 190 °C for 5 min. Simultaneous saccharification and co-fermentation of all the sugars (SScF) by B. coagulans resulted in a conversion of 80 % of available carbohydrates to optically pure lactic acid depending on the B. coagulans strain used as the microbial biocatalyst. Liquefaction of pretreated bagasse with cellulases before SScF (L + SScF) increased the productivity of lactic acid. These results show that B. coagulans is an effective biocatalyst for fermentation of all the sugars present in sweet sorghum juice and bagasse to optically pure lactic acid at high titer and productivity as feedstock for bio-based plastics.     

Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or Pyrocathechol

Reaction conditions for the synthesis of L-tyrosine or L-dopa from DL-serine and phenol or pyrocatechol were studied with intact cells of Erwinia herbicola (ATCC 21434) containing high tyrosine phenol lyase activity. The optimum pH for this reaction was around 8.0, and the optimum temperature range was between 37~40°C for the synthesis of L-tyrosine and between 15~25°C for that of L-dopa. Sodium sulfite and EDTA were added to protect the synthesized L-dopa from decomposition. As high concentrations of phenol or pyrocatechol denatured the enzyme, each substrate was fed to maintain the optimum concentration during incubation.

The reaction mixture (100 ml) containing 4.0 g of DL-serine, 1.0 g of phenol or 0.7 g of pyrocatechol, 0.5 g of ammonium acetate and the cells, was incubated. During incubation, phenol or pyrocatechol was fed at intervals to maintain the substrate at the initial concentration. 5.35 g of L-tyrosine or 5.10 g of L-dopa was synthesized in 100 ml of the reaction mixture.     

Inactivation of Complement by <Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase Preparations not Correlated with Enzyme Content

DELÁGE et al.¹ recently reported that L-asparaginase (L-A) in vitro inhibits human and guinea-pig whole serum complement and that L-A is a potent activator of C1. They therefore proposed that their results may “give a clue to the severe anaphylactic reactions noted in some patients on administration of the drug”. Because guinea-pig serum contains L-A in high quantities and Deláge et al. used reagents and methods that make their data difficult to interpret on a molecular basis, we have reinvestigated the effect of L-A preparations on human and guinea-pig complement.     

Some chromosome numbers of the CzechoslovakLeguminosae

Chromosome numbers of the Czechoslovak species of the genusLotus, from various localities have been determined. The paper includes the speciesLotus uliginosus Schkuhr,L.tenuis Waldst. etKit. andL. borbásii Ujhelyi.     

An <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine Transporter Isoform for Neurogenesis Facilitated by <Emphasis Type="SmallCaps">l</Emphasis>-Theanine

l-Theanine (=γ-glutamylethylamide) is an amino acid ingredient in green tea with a structural analogy to l-glutamine (l-GLN) rather than l-glutamic acid (l-GLU), with regards to the absence of a free carboxylic acid moiety from the gamma carbon position. l-theanine markedly inhibits [³H]l-GLN uptake without affecting [³H]l-GLU uptake in cultured neurons and astroglia. In neural progenitor cells with sustained exposure to l-theanine, upregulation of the l-GLN transporter isoform Slc38a1 expression and promotion of both proliferation and neuronal commitment are seen along with marked acceleration of the phosphorylation of mammalian target of rapamycin (mTOR) and relevant downstream proteins. Stable overexpression of Slc38a1 leads to promotion of cellular growth with facilitated neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells stably overexpressing Slc38a1, marked phosphorylation is seen with mTOR and downstream proteins in a fashion insensitive to the additional stimulation by l-theanine. The green tea amino acid l-theanine could thus elicit pharmacological actions to up-regulate Slc38a1 expression for activation of the mTOR signaling pathway required for cell growth together with accelerated neurogenesis after sustained exposure in undifferentiated neural progenitor cells. In this review, I summarize a novel pharmacological property of the green tea amino acid l-theanine for embryonic and adult neurogenesis with a focus on the endogenous amino acid analog l-GLN. A possible translational strategy is also discussed on the development of dietary supplements and nutraceuticals enriched of l-theanine for the prophylaxis of a variety of untoward impairments and malfunctions seen in patients with different neurodegenerative and/or neuropsychiatric disorders.     

Enhanced biocatalytic production of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. B-3 with in situ product removal using ion-exchange resin

Bioconversion of dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of l-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated l-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve l-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of dl-ATC for the in situ removal of l-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for l-cysteine and low adsorption for dl-ATC, which is a favorable option. With in situ addition of 60 g L^?1 resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L^?1 of dl-ATC was converted to l-cysteine with 90.4 % of yield and 28.6 mmol L^?1 h^?1 of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of l-cysteine was improved by 2.27-fold using ISPR method.     

<Emphasis Type="Italic">In vitro</Emphasis> Blastogenesis inhibited by <Emphasis Type="Italic">Erwinia carotovora</Emphasis><Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase

Escherichia coliL-asparaginase, an antileukaemic agent in man¹, inhibits in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴. We now report that another L-asparaginase preparation—from Erwinia carotovora—also possesses antileukaemic activity^5,6 and has a more pronounced immunosuppressive effect on in vitro blastogenesis than the E. coli enzyme.     

3,4-Dihydroxyphenylethylamine and 5-Hydroxytryptamine Metabolism in the Rat: Acidic Metabolites in Cisternal Cerebrospinal Fluid Before and After Giving Probenecid

3,4-Dihydroxyphenylethylamine (DA, dopamine) and 5-hydroxytryptamine (5-HT) turnover values were determined in freely moving male rats by measuring the rates of accumulation of the acidic metabolites of the above transmitters, i.e., 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in cisternal cerebrospinal fluid (CSF) samples after probenecid (200 mg/kg i.p.) administration. Determinations on samples before and after acid hydrolysis showed that the latter procedure was necessary for DA turnover determination. Thus whereas total (DOPAC + HVA) increased linearly with time after probenecid, free (DOPAC + HVA) did not. This was because the percentage of DOPAC + HVA in conjugated form increased with time. Determinations on a group of 28 rats during the dark (red light) period showed that cisternal amine metabolite concentrations before probenecid injection did not parallel turnover values. This was probably because individual differences in metabolite egress strongly affect the pre-probenecid values. The poor correlations between CSF tryptophan and 5-HT turnover suggested that differences of brain tryptophan concentration were not major determinants of differences of brain 5-HT metabolism within this group of normal rats. Considering that the rats were of similar weight and that the turnover values were all determined at approximately the same time of day, the three- to fourfold ranges of the turnover values are remarkable. The positive correlation between the DA and 5-HT turnovers of individual rats suggests the existence of common effects on DA and 5-HT turnover in normal rats.     

Levodopa-induced dyskinesia is associated with increased thyrotropin releasing hormone in the dorsal striatum of hemi-parkinsonian rats

Background

Dyskinesias associated with involuntary movements and painful muscle contractions are a common and severe complication of standard levodopa (L-DOPA, L-3,4-dihydroxyphenylalanine) therapy for Parkinson''s disease. Pathologic neuroplasticity leading to hyper-responsive dopamine receptor signaling in the sensorimotor striatum is thought to underlie this currently untreatable condition.

Methodology/Principal Findings

Quantitative real-time polymerase chain reaction (PCR) was employed to evaluate the molecular changes associated with L-DOPA-induced dyskinesias in Parkinson''s disease. With this technique, we determined that thyrotropin releasing hormone (TRH) was greatly increased in the dopamine-depleted striatum of hemi-parkinsonian rats that developed abnormal movements in response to L-DOPA therapy, relative to the levels measured in the contralateral non-dopamine-depleted striatum, and in the striatum of non-dyskinetic control rats. ProTRH immunostaining suggested that TRH peptide levels were almost absent in the dopamine-depleted striatum of control rats that did not develop dyskinesias, but in the dyskinetic rats, proTRH immunostaining was dramatically up-regulated in the striatum, particularly in the sensorimotor striatum. This up-regulation of TRH peptide affected striatal medium spiny neurons of both the direct and indirect pathways, as well as neurons in striosomes.

Conclusions/Significance

TRH is not known to be a key striatal neuromodulator, but intrastriatal injection of TRH in experimental animals can induce abnormal movements, apparently through increasing dopamine release. Our finding of a dramatic and selective up-regulation of TRH expression in the sensorimotor striatum of dyskinetic rat models suggests a TRH-mediated regulatory mechanism that may underlie the pathologic neuroplasticity driving dopamine hyper-responsivity in Parkinson''s disease.     

Enantiomer-Selective Photo-Induced Reaction of Protonated Tryptophan with Disaccharides in the Gas Phase

In order to investigate chemical evolution in interstellar molecular clouds, enantiomer-selective photo-induced chemical reactions between an amino acid and disaccharides in the gas phase were examined using a tandem mass spectrometer containing an electrospray ionization source and a cold ion trap. Ultraviolet photodissociation mass spectra of cold gas-phase noncovalent complexes of protonated tryptophan (Trp) enantiomers with disaccharides consisting of two d-glucose units, such as d-maltose or d-cellobiose, were obtained by photoexcitation of the indole ring of Trp. NH₂CHCOOH loss via cleavage of the C_α–C_β bond in Trp induced by hydrogen atom transfer from the NH₃ ⁺ group of a protonated Trp was observed in a noncovalent heterochiral H⁺(l-Trp)(d-maltose) complex. In contrast, a photo-induced chemical reaction forming the product ion with m/z 282 occurs in homochiral H⁺(d-Trp)(d-maltose). For d-cellobiose, both NH₂CHCOOH elimination and the m/z 282 product ion were observed, and no enantiomer-selective phenomena occurred. The m/z 282 product ion indicates that the photo-induced C-glycosylation, which links d-glucose residues to the indole moiety of Trp via a C–C bond, can occur in cold gas-phase noncovalent complexes, and its enantiomer-selectivity depends on the structure of the disaccharide.