An imaging approach for determining the mechanism of enhancement of intestinal absorption of an L-theanine supplement.

BACKGROUND & OBJECTIVE: Theanine (L-glutamylethylamide) contained in green tea is a functional food component that has been attracting attention due to its relaxation effect. It was shown that the ingredients added to the theanine formulations increased the absorption of theanine. If this mechanism can be elucidated, it would be possible to contribute to development of evidence-based formulations. In this study, we investigated the effect of ingredients in the formulations on the absorption of theanine in detail. MAIN METHODS: After oral administration of a mixture of theanine and additional components to Wistar rats the plasma concentration was determined by an HPLC and the pharmacokinetic parameters were calculated. In addition, a new system for evaluating intestinal blood flow was developed since the involvement of intestinal blood flow was considered as a factor that increased absorption of theanine. KEY FINDINGS: Plasma concentration of theanine increased significantly in the combined use group with eight ingredients containing piperine as compared with theanine only group. Piperine would increase theanine absorption by increased blood flow, not an inhibition of metabolism. We succeeded to develop a visual and quantitative system to evaluate the effect of these ingredients directly including piperine on the intestinal blood flow using indocyanine green while maintaining physiological conditions. SIGNIFICANCE: Increased intestinal blood flow by these ingredients including piperine enhanced the absorption of theanine. Other mechanisms may also be considered as the mechanism by which theanine absorption is increased in addition to increased blood flow.

Predictive value of 4-Hydroxyglutamate and miR-149-5p on eclampsia.

This research aimed at exploring the predictive value of 4-Hydroxyglutamate and miR-149-5p on eclampsia. Preeclampsia patients admitted to our hospital (n = 204), with 112 mild patients and 92 severe patients. Thereinto, pregnant women who underwent physical examination were regarded as a normal group (NG) (n = 100). Serum 4-Hydroxyglutamate levels and miR-149-5p in each group were detected. The serum 4-Hydroxyglutamate level in pregnant women in the NG was markedly lower than that in preeclampsia, while the miR-149-5p level was higher (p = 0.001). The serum 4-Hydroxyglutamate level in severe preeclampsia was higher than that in mild preeclampsia, while the miR-149-5p level was lower (p = 0.001). Partial thromboplastin time (APTT) and prothrombin time (PT) of preeclampsia patients were lower than those of the NG, while Fibrinogen (Fib) was higher (p = 0.001). With the aggravation of the condition of patients, PT, APTT decreased and Fib index increased. In preeclampsia patients, serum 4-Hydroxyglutamate was negatively correlated with PT and APTT, positively correlated with Fib content (p < 0.001); serum miR-149-5p was dramatically positively correlated with PT and APTT, negatively correlated with Fib content (p < 0.001). 4-Hydroxyglutamate and miR-149-5p were relevant to the occurrence time of preeclampsia; 4-Hydroxyglutamate, miR-149-5p and their combination could be used for preeclampsia diagnosis. According to the situation of newborn, they were divided into good and poor groups. The 4-Hydroxyglutamate level in the good group was lower than that in the poor group, while the miR-149-5p level was higher. The adverse prognosis of preeclampsia patients was predicted by 4-Hydroxyglutamate and miR-149-5p. 4-Hydroxyglutamate is highly expressed in preeclampsia, while miR-149-5p is low. Single and combined detection of 4-Hydroxyglutamate, miR-149-5p can be used for preeclampsia diagnosis and prediction.

One-carbon metabolites, B vitamins and associations with systemic inflammation and angiogenesis biomarkers among colorectal cancer patients: results from the ColoCare Study.

B vitamins involved in one-carbon metabolism have been implicated in the development of inflammation- and angiogenesis-related chronic diseases, such as colorectal cancer (CRC). Yet, the role of one-carbon metabolism in inflammation and angiogenesis among CRC patients remains unclear. The objective of this study was to investigate associations of components of one-carbon metabolism with inflammation and angiogenesis biomarkers among newly diagnosed CRC patients (n 238) in the prospective ColoCare Study, Heidelberg. We cross-sectionally analysed associations between twelve B vitamins and one-carbon metabolites and ten inflammation and angiogenesis biomarkers from pre-surgery serum samples using multivariable linear regression models. We further explored associations among novel biomarkers in these pathways with Spearman partial correlation analyses. We hypothesised that pyridoxal-5'-phosphate (PLP) is inversely associated with inflammatory biomarkers. We observed that PLP was inversely associated with C-reactive protein (CRP) (r -0·33, Plinear < 0·0001), serum amyloid A (SAA) (r -0·23, Plinear = 0·003), IL-6 (r -0·39, Plinear < 0·0001), IL-8 (r -0·20, Plinear = 0·02) and TNFα (r -0·12, Plinear = 0·045). Similar findings were observed for 5-methyl-tetrahydrofolate and CRP (r -0·14), SAA (r -0·14) and TNFα (r -0·15) among CRC patients. Folate catabolite acetyl-para-aminobenzoylglutamic acid (pABG) was positively correlated with IL-6 (r 0·27, Plinear < 0·0001), and pABG was positively correlated with IL-8 (r 0·21, Plinear < 0·0001), indicating higher folate utilisation during inflammation. Our data support the hypothesis of inverse associations between PLP and inflammatory biomarkers among CRC patients. A better understanding of the role and inter-relation of PLP and other one-carbon metabolites with inflammatory processes among colorectal carcinogenesis and prognosis could identify targets for future dietary guidance for CRC patients.

Plasma metabolites Xanthine, 4-Pyridoxate, and d-glutamic acid as novel potential biomarkers for pulmonary tuberculosis.

BACKGROUND: The lack of rapid and efficient diagnostic methods has been one of the most frustrating challenges in controlling the pulmonary tuberculosis (TB) epidemic. This study was aimed to identify novel non-invasive biomarkers for pulmonary TB. METHODS: The subjects in this study were divided into four groups: the pulmonary TB group, the community-acquired pneumonia (CAP) group, the lung cancer (LC) group, and the normal control (NC) group. Plasma small molecule metabolites were investigated in each group by using ultra-high performance liquid chromatography coupled with Q Exactive mass spectrometry. Multivariate statistical methods and bioinformatics were used to analyze the data. RESULTS: We identified three differential plasma metabolites such as, Xanthine, 4-Pyridoxate and d-glutamic acid in the pulmonary TB group, compared to the other groups (CAP, LC and NC). The pathway enrichment analysis indicated that the energy source in pulmonary TB was multi-center, which might be involved in maintaining the reproductive ability and virulence of Mycobacterium tuberculosis. CONCLUSION: The results suggested that Xanthine, 4-Pyridoxate, and d-glutamic acid may serve as potential biomarkers for pulmonary TB. The present study provides experimental basis for developing potential biomarkers of pulmonary TB.

Monosodium Glutamate in the Diet Does Not Raise Brain Glutamate Concentrations or Disrupt Brain Functions.

The non-essential amino acid glutamate participates in numerous metabolic pathways in the body. It also performs important physiologic functions, which include a sensory role as one of the basic tastes (as monosodium glutamate [MSG]), and a role in neuronal function as the dominant excitatory neurotransmitter in the central nervous system. Its pleasant taste (as MSG) has led to its inclusion as a flavoring agent in foods for centuries. Glutamate's neurotransmitter role was discovered only in the last 60 years. Its inclusion in foods has necessitated its safety evaluation, which has raised concerns about its transfer into the blood ultimately increasing brain glutamate levels, thereby causing functional disruptions because it is a neurotransmitter. This concern, originally raised almost 50 years ago, has led to an extensive series of scientific studies to examine this issue, conducted primarily in rodents, non-human primates, and humans. The key findings have been that (a) the ingestion of MSG in the diet does not produce appreciable increases in glutamate concentrations in blood, except when given experimentally in amounts vastly in excess of normal intake levels; and (b) the blood-brain barrier effectively restricts the passage of glutamate from the blood into the brain, such that brain glutamate levels only rise when blood glutamate concentrations are raised experimentally via non-physiologic means. These and related discoveries explain why the ingestion of MSG in the diet does not lead to an increase in brain glutamate concentrations, and thus does not produce functional disruptions in brain. This article briefly summarizes key experimental findings that evaluate whether MSG in the diet poses a threat to brain function.

High and individually variable enzymatic activity precludes accurate determination of pemetrexed, methotrexate and their polyglutamate metabolite concentrations in plasma.

Most drugs are metabolized in the human body. Therefore, it is essential for therapeutic drug monitoring studies to also take into account the concentrations of drug metabolites. One of the possible metabolic activities on drugs such as pemetrexed or methotrexate is (poly)glutamation. Here, we report on a series of experiments that we performed to investigate the stability of polyglutamate metabolites in plasma. Removal of glutamate residues from pemetrexed polyglutamate by most likely proteases in human plasma is influenced by temperature as it is observed at 25°C and even more strongly at 37°C, but not at 4°C. The observed protease activity is highly variable among patients; in approximately 15-20% of the patients tested it is not observed, whereas in other individuals the activity is so extensive that after 10min, more than 50% of spiked polyglutamated pemetrexed is degraded at room temperature (5-10% of the tested individuals). Similar observations also pertain to methotrexate polyglutamates. These observations do not extend to pemetrexed and methotrexate themselves which are unaffected by this activity. Due to the considerable and, among individuals, variable protease activities on polyglutamated drug metabolites in plasma, these metabolites are virtually impossible to quantify if no precautions are taken.

Dodecafluoropentane Improves Neurological Function Following Anterior Ischemic Stroke.

Dodecafluoropentane emulsion (DDFPe), an advanced oxygen transport drug, given IV at 90-min intervals maintains viability in the penumbra during cerebral ischemia in the standard rabbit anterior stroke model (STND). This study investigated shortened dosage schedules of DDFPe in nonstandard posterior (NSTND) strokes following occlusions of the posterior cerebral arteries. DDFPe given at shortened schedules of 30 or 60-min injection intervals will reduce neurological deficits, percent stroke volume (%SV), and serum glutamate levels in NSTND ischemic strokes. New Zealand White rabbits (N = 26) were randomly placed into three groups: A (n = 9) controls given saline injections every 60 min, B (n = 9) 2 % DDFPe given IV every 30 min, and C (n = 8) DDFPe every 60 min. Injections began 1 h after embolization. Groups were subdivided into STND and NSTND based on angiographically verified embolization of the cerebral arteries. Neurological assessments and blood samples were done at 0.5-1-h intervals. Rabbits were euthanized at 7 h following embolization. Stained brain slices were measured for %SV. The 30 and 60-min subgroups did not differ and were combined as DDFPe-STND or DDFPe-NSTND groups. In the DDFPe-STND stroke group, the %SV, neurological assessment scores (NAS), and serum glutamate were decreased vs. STND controls (p = 0.0016, 0.008, and 0.016, respectively). In the DDFPe-NSTND stroke group, %SV, NAS, and serum glutamate did not differ statistically compared to NSTND controls (p = 0.82, 0.097, and 0.06, respectively). More frequent dosage schedules provided no additional improvement. In anterior strokes, DDFPe improves recovery but not in the more severe NSTND strokes.

A new quantification method for assessing plasma concentrations of pemetrexed and its polyglutamate metabolites.

Currently no quantification method exists for potentially therapeutically relevant polyglutamate metabolites of the drug pemetrexed which is used for the treatment of lung carcinoma patients. We developed and tested an LC-MS/MS-based analytical assay that uses isotope-labeled internal standards to quantify pemetrexed and its (poly)glutamate metabolites in clinical human plasma samples of lung carcinoma patients. UHPLC chromatography and triple quadrupole mass spectrometry showed an LLOQ of 0.2nmol/L for pemetrexed and an LLOQ of 0.5nmol/L for the two metabolites (one glutamate and two glutamate moieties covalently bound to the pemetrexed molecule, for which no other quantification methods have previously been published). The recoveries for PMTX and its metabolites ranged between 30% and 67%. Precision and accuracy at a concentration of 20nmol/L for all four analytes was well below 15% CV. The precision (RSD) in the biological replicates of the separate days (within-run precision) as well as the reproducibility over several days (between-run precision), tested in the range of 5-250nmol/L, were all below 15%. Autosampler, benchtop and freeze-thaw cycle stability of the analytes was also demonstrated. To illustrate the new assay in a relevant biological context, concentrations of pemetrexed and the two metabolites were quantified in plasma samples of lung carcinoma patients treated with pemetrexed. The assay is straightforward, relatively easy to perform, and has potential for use in therapeutic drug monitoring in non-small cell lung carcinoma patients.

Obesity changes the human gut mycobiome.

The human intestine is home to a diverse range of bacterial and fungal species, forming an ecological community that contributes to normal physiology and disease susceptibility. Here, the fungal microbiota (mycobiome) in obese and non-obese subjects was characterized using Internal Transcribed Spacer (ITS)-based sequencing. The results demonstrate that obese patients could be discriminated by their specific fungal composition, which also distinguished metabolically "healthy" from "unhealthy" obesity. Clusters according to genus abundance co-segregated with body fatness, fasting triglycerides and HDL-cholesterol. A preliminary link to metabolites such as hexadecanedioic acid, caproic acid and N-acetyl-L-glutamic acid was also found. Mucor racemosus and M. fuscus were the species more represented in non-obese subjects compared to obese counterparts. Interestingly, the decreased relative abundance of the Mucor genus in obese subjects was reversible upon weight loss. Collectively, these findings suggest that manipulation of gut mycobiome communities might be a novel target in the treatment of obesity.

Thrombolytic treatment decreases glutamate/GABA ratio in serum during acute ischaemic stroke: a pilot study.

There is no information about possible effect of recombinant tissue plasminogen activator (rtPA) therapy on excitotoxic/neuroprotective amino acids during acute phase of ischaemic stroke (IS). Our purpose was to evaluate iv thrombolytic treatment on glutamate (Glu) and gamma-aminobutyric acid (GABA) serum levels during acute IS. Eleven thrombolytic (rtPA group) and 12 non-thrombolytic (non-rtPA group) patients with acute IS were enrolled. The serum samples were obtained at three time points for rtPA group (time point 0: first to fourth hour of stroke; time point 1: immediately after rtPA administration; time point 2: on days 5-7 from stroke onset). The remaining patients had blood collection at two time points: time point 1: 5(th)-10(th) hour of stroke and time point 2: on days 5-7 of stroke. Glutamate and GABA were determined by the automated ion-exchange chromatography using Amino Acids Analyser (AAA 400) by INGOS Corp., Praha, Czech Republic. The statistically significant elevation of GABA serum level was noticed directly after thrombolysis (time point 1) in comparison to the corresponding time point in non-rtPA group [0.016 (0.002-0.032) µM/ml vs 0.001 (0.001-0.004) µM/ml for rtPA vs non-rtPA groups, respectively, median (first to third quartile), P < 0.05]. At the same time point, the Glu/GABA ratio was significantly decreased in rtPA group (P < 0.05) suggesting the decrease of excitotoxicity biomarkers in the blood after thrombolysis. Considering the beneficial effect of GABA receptor agonists, the elevation of GABA by rtPA should bring an additional positive features of thrombolytic treatment.

Periodic hypokalaemic polymyopathy in Burmese and closely related cats: a review including the latest genetic data.

GLOBAL IMPORTANCE: Hypokalaemic polymyopathy is a genetic disease of Burmese cats that has been encountered in Australasia, Europe and South Africa. CLINICAL FEATURES: Affected cats usually present with signs of muscle weakness and muscle pain in the first year of life. Although certain clinical features, such as ventroflexion of the head and neck, are especially characteristic, some cats do not display these signs. Usually weakness is periodic or episodic, but occasionally it is incessant. DIAGNOSTIC CHALLENGES: In the past, diagnosis was problematic in that clinical signs and a lowered serum potassium concentration were not always observed synchronously. This necessitated serial serum potassium concentration determinations, testing of serum creatine kinase activity and exclusion of other potential causes of muscle disease in cats (including muscular dystrophies, Toxoplasma myositis, immune-mediated polymyositis, organophosphorus intoxication and envenomations). Signs in affected cats often waxed and waned, possibly in response to changes in dietary factors and stress, and some cats could apparently 'grow out of' the condition. RECENT ADVANCES AND FUTURE PROSPECTS: Recent molecular genetics research has identified a single nonsense mutation in the gene (WNK4) coding for lysine-deficient 4 protein kinase, an enzyme present primarily in the distal nephron. The underlying pathomechanism in affected cats is therefore likely to be a potassium wasting nephropathy, as this enzyme is involved in complex sodium/potassium exchange mechanisms in the kidney. Additional functional characterisation of the condition is warranted to define precisely how, why and when the serum potassium concentration declines. The diagnosis of Burmese hypokalaemia is now straightforward, as an inexpensive PCR test can identify affected homozygous individuals, as well as carriers. The elimination of this condition from the Burmese breed, and also from pedigree cats infused with Burmese lines, such as the Bombay, Tonkinese and Tiffanie breeds, should therefore be possible.

Challenges in optimizing sample preparation and LC-MS/MS conditions for the analysis of carglumic acid, an N-acetyl glutamate derivative in human plasma.

This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed-phase solid-phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic-lipophilic balance, and mixed-mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed-mode anion exchange (~ 50%) cartridge, compared to other sorbents from 100 µL plasma sample. Complete analytical separation of carglumic acid and carglumic acid-13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 × 4.6 mm, 5 µm) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) - 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor → product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00-6000 ng/mL (r(2) ≥ 0.9987). Matrix effect was evaluated in terms of IS-normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra- and inter-batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject.

Milk production, plasma metabolite profiles and mammary arterial-venous differences of milk precursors in early lactation cows milked at different frequencies by an automatic milking system.

The objective of this study was to clarify the effect of different milking frequencies under an automatic milking system (AMS) on milk yield, plasma metabolite profiles and mammary arterial-venous (A-V) differences of milk precursors by mammary tissues in early lactation cows. Twelve Holstein cows were divided into two and four times milking frequency treatments by AMS after calving to 50 days postpartum. Cows were given a partial mixed ration ad libitum and a concentrate diet at every milking. Dry matter intake increased similarly in both treatments with advancing postpartum days. Milk yield was greater (P < 0.001) by 25% with four times milking, but milk composition was not affected by milking frequency. Body weight change was also not affected by milking frequency. Arterial concentrations of glucose and glutamate were lower (P < 0.05) for four times milking frequency. However, arterial concentration of nonesterified fatty acids did not differ between treatments. Although mammary A-V differences of plasma concentration for most milk precursors did not differ between treatments, estimated plasma flow was higher (P < 0.05) for four times milking frequency. These results indicate that higher milking frequency may increase mammary uptake of milk precursors, whereas may not affect the extent of fat mobilization of early lactating cows from day 20 postpartum onward.

Preclinical analyses and phase I evaluation of LY2603618 administered in combination with pemetrexed and cisplatin in patients with advanced cancer.

LY2603618 is an inhibitor of checkpoint kinase 1 (CHK1), an important regulator of the DNA damage checkpoints. Preclinical experiments analyzed NCI-H2122 and NCI-H441 NSCLC cell lines and in vitro/in vivo models treated with pemetrexed and LY2603618 to provide rationale for evaluating this combination in a clinical setting. Combination treatment of LY2603618 with pemetrexed arrested DNA synthesis following initiation of S-phase in cells. Experiments with tumor-bearing mice administered the combination of LY2603618 and pemetrexed demonstrated a significant increase of growth inhibition of NCI-H2122 (H2122) and NCI-H441 (H441) xenograft tumors. These data informed the clinical assessment of LY2603618 in a seamless phase I/II study, which administered pemetrexed (500 mg/m(2)) and cisplatin (75 mg/m(2)) and escalating doses of LY2603618: 130-275 mg. Patients were assessed for safety, toxicity, and pharmacokinetics. In phase I, 14 patients were enrolled, and the most frequently reported adverse events included fatigue, nausea, pyrexia, neutropenia, and vomiting. No DLTs were reported at the tested doses. The systemic exposure of LY2603618 increased in a dose-dependent manner. Pharmacokinetic parameters that correlate with the maximal pharmacodynamic effect in nonclinical xenograft models were achieved at doses ≥240 mg. The pharmacokinetics of LY2603618, pemetrexed, and cisplatin were not altered when used in combination. Two patients achieved a confirmed partial response (both non-small cell lung cancer), and 8 patients had stable disease. LY2603618 administered in combination with pemetrexed and cisplatin demonstrated an acceptable safety profile. The recommended phase II dose of LY2603618 was 275 mg.

Effect of L-theanine on sensorimotor gating in healthy human subjects.

AIM: l-Theanine (N-ethyl-l-glutamine) is an amino acid uniquely found in green tea. Growing evidence has suggested the possible effects of l-theanine on cognition. Previously, we found that l-theanine attenuates MK-801-induced deficit in prepulse inhibition (PPI) in mice. In this study, we examined the effect of l-theanine in increasing the PPI in healthy humans. METHODS: The subjects were 14 healthy adults who underwent PPI testing as a measure of sensorimotor gating 90 min after an oral intake of l-theanine (0, 200, 400, or 600 mg). PPI tests were done by examiners who were blind to the dose. RESULTS: The administration of 200 mg of l-theanine and that of 400 mg, but not 600 mg, significantly increased the % PPI compared to the baseline (0 mg). There was no significant relation between the dose of l-theanine and the startle magnitude or the habituation of startle response. The plasma concentrations of l-theanine correlated with the dose of l-theanine. CONCLUSION: The observed effect with 200-400 mg of l-theanine on PPI suggested that l-theanine at a particular dose range increases sensorimotor gating in humans.

Folate determination in human health: UPLC-MS/MS is the emerging methodology of choice.

This Perspective provides a brief description of the essential role that folates play in human health, together with an overview of the various analytical methods that have been used for quantitation of folates in human populations over the past few decades. Essentially, folate methodology has evolved from microbiological assay-based, to binding-based technology and, more recently, to separation-based methodology. Separation-based methods initially used traditional LC in conjunction with various detection techniques, with the most recent methods utilizing UPLC-MS/MS. Current UPLC methods offer exceptional speed, sensitivity and quantitation ability for the monoglutamate folate isoforms. It appears that the only limitation to properly quantifying all folates as polyglutamates, some 40-50 species, is the current lack of corresponding stable-isotope standards. Clearly, UPLC-MS/MS is emerging as the 'method of choice' for the determination of folates, whether in support of basic research, clinical investigations or population studies.

Identification of honokiol metabolites in rats by the method of stable isotope cluster technique and ultra-high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

Honokiol, a natural molecule isolated from Magnolia officinalis Rehd. et Wils., is widely known as an antitumor agent. In present work, an analysis of in vivo biotransformation and metabolites of honokiol has been performed by a combined method based on stable isotope cluster technique with honokiol-[(13)C6]-labeled and ultra-high performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry (UHPLC/Q-TOF-MS). The metabolites could be easily identified by the determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to that between isotope-labeled and non-isotope-labeled honokiol. A total of eighteen metabolites were detected and tentatively identified, fourteen of which were reported for the first time. The results indicated that the main metabolic pathways of honokiol in rats were hydroxylation, methylation, sulfation and glucuronidation. This study provided the first essential information on biotransformation and metabolites of honokiol in rats, which was very useful for further pharmacological and clinical studies of honokiol as a potent drug candidate.

Folate catabolites in spot urine as non-invasive biomarkers of folate status during habitual intake and folic acid supplementation.

BACKGROUND: Folate status, as reflected by red blood cell (RCF) and plasma folates (PF), is related to health and disease risk. Folate degradation products para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (apABG) in 24 hour urine have recently been shown to correlate with blood folate. AIM: Since blood sampling and collection of 24 hour urine are cumbersome, we investigated whether the determination of urinary folate catabolites in fasted spot urine is a suitable non-invasive biomarker for folate status in subjects before and during folic acid supplementation. STUDY DESIGN AND METHODS: Immediate effects of oral folic acid bolus intake on urinary folate catabolites were assessed in a short-term pre-study. In the main study we included 53 healthy men. Of these, 29 were selected for a 12 week folic acid supplementation (400 µg). Blood, 24 hour and spot urine were collected at baseline and after 6 and 12 weeks and PF, RCF, urinary apABG and pABG were determined. RESULTS: Intake of a 400 µg folic acid bolus resulted in immediate increase of urinary catabolites. In the main study pABG and apABG concentrations in spot urine correlated well with their excretion in 24 hour urine. In healthy men consuming habitual diet, pABG showed closer correlation with PF (rs = 0.676) and RCF (rs = 0.649) than apABG (rs = 0.264, ns and 0.543). Supplementation led to significantly increased folate in plasma and red cells as well as elevated urinary folate catabolites, while only pABG correlated significantly with PF (rs = 0.574) after 12 weeks. CONCLUSION: Quantification of folate catabolites in fasted spot urine seems suitable as a non-invasive alternative to blood or 24 hour urine analysis for evaluation of folate status in populations consuming habitual diet. In non-steady-state conditions (folic acid supplementation) correlations between folate marker (RCF, PF, urinary catabolites) decrease due to differing kinetics.

Brain efflux index to investigate the influence of active efflux on brain distribution of pemetrexed and methotrexate.

Antifolates, in particular methotrexate (MTX), have been widely used in the treatment of primary and secondary tumors of the central nervous system (CNS). Pemetrexed (PMX) is a novel antifolate that also exhibits potent antitumor activity against CNS malignancies. Studies have shown that brain distribution of both antifolates is significantly restricted, possible due to active efflux transport at the blood-brain barrier (BBB). This study characterizes the brain-to-blood transport of PMX and MTX and examines the role of several efflux transporters in brain distribution of the antifolates by use of the intracerebral microinjection technique (brain efflux index). The results from this study show that both PMX and MTX undergo saturable efflux transport across the BBB, with elimination half-lives of approximately 39 minutes and 29 minutes, respectively. Of the various efflux transporters this study investigated, multidrug resistance-associated protein 2 (Mrp2) does not play an important role in the brain distribution of the two antifolate drugs. Interestingly, breast-cancer resistance protein (Bcrp) makes a significant contribution to the brain elimination of MTX but not PMX. In addition, the brain-to-blood transport of both antifolates was inhibited by probenecid and benzylpenicillin, suggesting the involvement of organic anion transporters in the efflux of these compounds from the brain, with organic anion transporter 3 (Oat3) being a possibility. Our results suggest that one of the underlying mechanisms behind the limited brain distribution of PMX and MTX is active efflux transport processes at the BBB, including a benzylpenicillin-sensitive transport system and/or the active transporter Bcrp.

Pharmacokinetics and efficacy of pemetrexed in patients with brain or leptomeningeal metastases.

Brain metastases (BM) and leptomeningeal metastases (LM) are devastating neurologic complications. Pemetrexed is a multi-targeted anti-folate agent approved for treatment of nonsquamous non-small cell lung cancer but has anti-tumor activity in other solid tumors. We performed two trials using pemetrexed in patients with BM and LM to assess CSF penetration and anti-tumor activity. Patients were treated with intravenous pemetrexed at doses of 500 (n = 3), 750 (n = 3), 900 (n = 12) or 1,050 mg/m(2) (n = 3) every 3 weeks. Neuro-imaging was done every 6 weeks. Matched CSF and plasma samples were obtained serially from three patients with Ommaya reservoirs; the remaining patients had a single paired collection. Twenty-one patients (15 women and six men) with median age of 50 years and median KPS of 90 were treated. Primary tumors included breast (13), lung (4), colorectal (1), endometrial (1), esophageal (1) and pinealoblastoma (1). Nine patients had prior whole brain RT and median number of prior chemotherapies was two including prior methotrexate in four patients. Median pemetrexed doses administered was three (range 1-14). Responses included one partial response, ten stable disease and ten progressive disease. Median time to progression and survival was 2.7 and 7.3 months; PFS six was 22 %. No major toxicities were seen. Pemetrexed distributed from the plasma to the CSF within 1-4 h with the resulting CSF concentrations < 5 % of plasma. Pemetrexed was tolerated in solid tumor patients with CNS metastases. Limited anti-tumor activity was seen, which might have been due to low CSF concentrations, although some patients displayed prolonged benefit.