Identification and quantification of (poly)phenol and methylxanthine metabolites in human cerebrospinal fluid: evidence of their ability to cross the BBB.

Increasing evidence suggests that dietary (poly)phenols and methylxanthines have neuroprotective effects; however, little is known about whether they can cross the blood-brain barrier (BBB) and exert direct effects on the brain. We investigated the presence of (poly)phenol and methylxanthine metabolites in plasma and cerebrospinal fluid (CSF) from 90 individuals at risk of dementia using liquid chromatography-mass spectrometry and predicted their mechanism of transport across the BBB using in silico modelling techniques. A total of 123 and 127 metabolites were detected in CSF and plasma, respectively. In silico analysis suggests that 5 of the 20 metabolites quantified in CSF can cross the BBB by passive diffusion, while at least 9 metabolites require the aid of cell transporters to cross the BBB. Our results showed that (poly)phenols and methylxanthines are bioavailable, can cross the BBB via passive diffusion or transport carriers, and can reach brain tissues to exert neuroprotective effects.

Sex and race differences of cerebrospinal fluid metabolites in healthy individuals.

INTRODUCTION: Analyses of cerebrospinal fluid (CSF) metabolites in large, healthy samples have been limited and potential demographic moderators of brain metabolism are largely unknown. OBJECTIVE: Our objective in this study was to examine sex and race differences in 33 CSF metabolites within a sample of 129 healthy individuals (37 African American women, 29 white women, 38 African American men, and 25 white men). METHODS: CSF metabolites were measured with a targeted electrochemistry-based metabolomics platform. Sex and race differences were quantified with both univariate and multivariate analyses. Type I error was controlled for by using a Bonferroni adjustment (0.05/33 = .0015). RESULTS: Multivariate Canonical Variate Analysis (CVA) of the 33 metabolites showed correct classification of sex at an average rate of 80.6% and correct classification of race at an average rate of 88.4%. Univariate analyses revealed that men had significantly higher concentrations of cysteine (p < 0.0001), uric acid (p < 0.0001), and N-acetylserotonin (p = 0.049), while women had significantly higher concentrations of 5-hydroxyindoleacetic acid (5-HIAA) (p = 0.001). African American participants had significantly higher concentrations of 3-hydroxykynurenine (p = 0.018), while white participants had significantly higher concentrations of kynurenine (p < 0.0001), indoleacetic acid (p < 0.0001), xanthine (p = 0.001), alpha-tocopherol (p = 0.007), cysteine (p = 0.029), melatonin (p = 0.036), and 7-methylxanthine (p = 0.037). After the Bonferroni adjustment, the effects for cysteine, uric acid, and 5-HIAA were still significant from the analysis of sex differences and kynurenine and indoleacetic acid were still significant from the analysis of race differences. CONCLUSION: Several of the metabolites assayed in this study have been associated with mental health disorders and neurological diseases. Our data provide some novel information regarding normal variations by sex and race in CSF metabolite levels within the tryptophan, tyrosine and purine pathways, which may help to enhance our understanding of mechanisms underlying sex and race differences and potentially prove useful in the future treatment of disease.

Association of caffeine and related analytes with resistance to Parkinson disease among LRRK2 mutation carriers: A metabolomic study.

OBJECTIVE: To identify markers of resistance to developing Parkinson disease (PD) among LRRK2 mutation carriers (LRRK2+), we carried out metabolomic profiling in individuals with PD and unaffected controls (UC), with and without the LRRK2 mutation. METHODS: Plasma from 368 patients with PD and UC in the LRRK2 Cohort Consortium (LCC), comprising 118 LRRK2+/PD+, 115 LRRK2+/UC, 70 LRRK2-/PD+, and 65 LRRK2-/UC, and CSF available from 68 of them, were analyzed by liquid chromatography with mass spectrometry. For 282 analytes quantified in plasma and CSF, we assessed differences among the 4 groups and interactions between LRRK2 and PD status, using analysis of covariance models adjusted by age, study site cohort, and sex, with p value corrections for multiple comparisons. RESULTS: Plasma caffeine concentration was lower in patients with PD vs UC (p < 0.001), more so among LRRK2+ carriers (by 76%) than among LRRK2- participants (by 31%), with significant interaction between LRRK2 and PD status (p = 0.005). Similar results were found for caffeine metabolites (paraxanthine, theophylline, 1-methylxanthine) and a nonxanthine marker of coffee consumption (trigonelline) in plasma, and in the subset of corresponding CSF samples. Dietary caffeine was also lower in LRRK2+/PD+ compared to LRRK2+/UC with significant interaction effect with the LRRK2+ mutation (p < 0.001). CONCLUSIONS: Metabolomic analyses of the LCC samples identified caffeine, its demethylation metabolites, and trigonelline as prominent markers of resistance to PD linked to pathogenic LRRK2 mutations, more so than to idiopathic PD. Because these analytes are known both as correlates of coffee consumption and as neuroprotectants in animal PD models, the findings may reflect their avoidance by those predisposed to develop PD or their protective effects among LRRK2 mutation carriers.

HPLC method for measurement of purine nucleotide degradation products in cerebrospinal fluid.

We describe a convenient method for the separation and quantification of xanthine, hypoxanthine, and uric acid in 20 microL of cerebrospinal fluid (CSF) with use of HPLC and ultraviolet detection. The analysis is performed on a Sepharon SGX C18 column and the elution system consists of potassium phosphate buffer, pH 5.1, with 20 mL/L methanol. The lower limit of detection was 4 pmol for hypoxanthine and xanthine and 6 pmol for uric acid. Analytical recoveries of purine metabolites ranged from 98.6% to 102.9%. The intra- and interassay CVs were <3%. The applicability of the method is illustrated with the determination of micromolar concentrations of xanthine, hypoxanthine, and uric acid in CSF samples obtained from 113 patients with various neurological disorders.

Xanthine analysis in biological fluids by capillary electrophoresis.

Xanthine, a precursor of uric acid, is measured here in serum, urine, and cerebrospinal fluids by capillary electrophoresis (CE) after deproteinization with acetonitrile. The migration time is about 7.5 min with a minimum detection limit of 0.4 mg/l. Different purines and pyrimidines did not interfere with the determination. The method demonstrates the suitability of the CE for determination of small molecules present in a complex matrix at levels of ca. 1mg/l. It also demonstrates that acetonitrile deproteinization is a simple and effective method for preparing samples for CE, allowing a large volume to be introduced into the capillary.

Concentration of purine compounds in the cerebrospinal fluid of infants suffering from sepsis, convulsions and hydrocephalus.

Catabolites of purine nucleotides were measured in the cerebrospinal fluid (CSF) of newborn infants with sepsis, seizures and hydrocephalus using isocratic reversed-phase HPLC. The inosine levels in the CSF of the infants with any of the illnesses were significantly higher when compared with the controls. There was a tendency for hypoxanthine levels to be higher in the group of children with hydrocephalus. No significant differences in the concentrations of xanthine, adenine and uric acid were found. The inosine concentration in the CSF is proposed to be a more sensitive indicator of brain injury than the levels of other CSF purines. The levels of all purine metabolites measured in the CSF showed large individual variations. The ratio between hypoxanthine (as an indicator of ATP breakdown) and uric acid (as a scavenger of oxygen free radicals) concentration is proposed as a new criterion to be used in the evaluation of brain injury.

Effects of asphyxia on the fetal lamb brain.

OBJECTIVE: Our purpose was to study the effect of fetal asphyxia on the release of hypoxanthine and xanthine in cerebrospinal fluid and on brain histologic characteristics. STUDY DESIGN: In seven fetal lambs (3 to 5 days after surgery, gestational age 124.3 +/- 2.6 days) asphyxia was induced by restriction of uterine blood flow. RESULTS: Fetal pH and base excess were reduced to 6.99 +/- 0.02 and -17.6 +/- 0.9 mmol/L, respectively. Cerebral blood flow increased during asphyxia and returned to normal in the recovery phase. Maximum concentrations of cerebrospinal fluid hypoxanthine and xanthine were reached in the normoxemic recovery phase. This high level of substrates during normoxemia facilitates oxygen free radical formation and may thus aggravate postasphyctic brain damage. Histologic evaluation of the brain 3 days after the insult showed a variable degree of edema. Coagulative neuronal changes, characteristic of irreversible cell death, were only occasionally detected. These changes were most obvious in the Purkinje cells of the cerebellum. CONCLUSIONS: Fetal asphyxia induced by uterine blood flow restriction is associated with high levels of cerebrospinal fluid hypoxanthine and xanthine in the recovery phase. Microscopically detectable brain damage, although not extensive, is mainly located in the cerebellum.

Concentrations of nucleotides, nucleosides, purine bases and urate in cerebrospinal fluid of children with meningitis.

The release of agents mediating inflammation in meningitis may bring about neuronal hypoxia, under which circumstances ATP concentrations decrease and its degradation products increase and are released into the cerebrospinal fluid. In this study of alterations in neuronal energy metabolism in meningitis, AMP, IMP, inosine, adenosine, guanosine, adenine, guanine, hypoxanthine, xanthine and urate were determined by high performance liquid chromatography in the cerebrospinal fluid of 54 children aged between 1 month and 13 years suffering from meningitis (25 viral, 24 bacterial and 5 tuberculous cases) and 63 controls. Compared to the controls, patients with viral meningitis exhibited high concentrations of IMP, adenosine, guanosine, adenine, guanine and xanthine; patients with bacterial meningitis exhibited high concentrations of IMP, inosine, guanosine, adenosine, hypoxanthine, xanthine and urate; and patients with tuberculous meningitis exhibited high concentrations of AMP, guanosine, xanthine and urate. Viral and bacterial cases did not differ significantly for any of the metabolites studied. AMP and urate concentrations were significantly higher in patients with tuberculous cases compared with viral or bacterial meningitis cases.

Indicators of hypoxia in cerebrospinal fluid of hydrocephalic children with suspected shunt malfunction.

We used high performance liquid chromatography to determine the concentration of purine metabolites in the cerebrospinal fluid of three hydrocephalic children with a history of shunt malfunction. Hypoxanthine and xanthine levels were high in comparison with controls. We consider these purines to be valuable indicators of disturbance of neuronal metabolism following the sustained rise in intracranial pressure caused by shunt valve malfunction.

Transport of hypoxanthine from plasma to cerebrospinal fluid and vitreous humor in newborn pigs.

To determine whether an elevated level of hypoxanthine in cerebrospinal fluid or vitreous humor might reflect a high plasma hypoxanthine concentration, or whether it necessarily represents local tissue hypoxia, we infused hypoxanthine intravenously to normoxemic and normotensive piglets (n = 6). Hypoxanthine was measured in different body fluids using HPLC. During the 8 hours of infusion hypoxanthine increased in plasma (from 30 +/- 6 mumol/l (mean +/- SD) before the infusion to 68 +/- 20 mumol/l at the end of the infusion, p < 0.01), cerebrospinal fluid (CSF) (19 +/- 8 to 43 +/- 9 mumol/l, p < 0.05) and vitreous humor (15 +/- 5 to 30 +/- 6 mumol/l, p < 0.05). After infusion, hypoxanthine values in all three fluids were similar to those seen in pigs after severe hypoxia. Hypoxanthine in vitreous humor and plasma were significantly correlated (r = 0.80, 95% confidence interval 0.47-0.93, p < 0.001). Urinary excretion of hypoxanthine increased almost 40 times from 0.12 +/- 0.14 to 4.6 +/- 2.9 mumol/kg/h indicating that renal excretion of hypoxanthine is not achieved just by passive filtration. We conclude that in newborn piglets hypoxanthine can pass from plasma to CSF and vitreous humor. Thus an increased CSF hypoxanthine concentration is not definite proof that significant cerebral hypoxia has occurred.

The urate and xanthine concentrations in the cerebrospinal fluid in patients with vascular dementia of the Binswanger type, Alzheimer type dementia, and Parkinson's disease.

We determined the urate and xanthine concentrations in the cerebrospinal fluid (CSF) in patients with vascular dementia of the Binswanger type (VDBT), Alzheimer type dementia (ATD), and Parkinson's disease (PD). We found that the urate concentration was significantly increased in VDBT patients, but significantly decreased in ATD patients compared with controls. The ratio of the concentrations of uric acid (UCSF) to xanthine (XCSF) in the CSF (UCSF/XCSF) had a significant correlation with the ratio of the UCSF to the urate concentration in serum (U(serum)) (UCSF/U(serum)) in ATD and PD, whereas UCSF/U(serum) increased independently of UCSF/XCSF in VDBT. We concluded that the significant increase in the urate concentration in VDBT is mainly due to an impairment of the blood-brain barrier (BBB), and its significant reduction in ATD may reflect impaired brain metabolism.

The effect of age on concentrations of monoamines, amino acids, and their related substances in the cerebrospinal fluid.

We studied age-related changes in the concentrations of monoamines, amino acids, and their related substances in the cerebrospinal fluid on 144 neurologically normal subjects. The concentrations of tyrosine, 3-O-methyldopa, dopamine (total), norepinephrine (total), homovanillic acid, p-hydroxyphenylacetic acid, and 5-hydroxytryptophan increased significantly with age (p < 0.05), and the concentration of 3.4-dihydroxyphenylacetic acid displayed a non-significant trend to decrease, whereas concentrations of other monoamine precursors and metabolites were unchanged. We found the significant positive correlations between the concentrations of HVA and 5-HIAA (p < 0.001), between tyrosine and tryptophan (p < 0.001), and between tyrosine and 3-O-methyldopa (p < 0.001). The concentrations of asparagine, glycine, taurine, and alanine increased significantly with age (p < 0.05), while glutamine, arginine, and threonine concentrations did not change with age. The aspartate, glutamate, and GABA concentrations displayed the non-significant trends to decrease in the elderly subjects. The concentrations of aspartate, glutamate, and GABA had mutually significant positive correlations (p < 0.05), but had significant negative correlations with the concentrations of some neutral amino acids. The urate and xanthine concentrations increased significantly with age (p < 0.01). These findings suggest that the concentrations of monoamine and amino acid transmitters and their related compounds in the cerebrospinal fluid reflect age-related changes in the synthesis, release, and reuptake mechanisms of the transmitters and their transport mechanisms across the blood-brain barrier.

Purine metabolites and pyrimidine bases in cerebrospinal fluid of children with simple febrile seizures.

Adenosine monophosphate, inosine monophosphate, inosine, adenosine, guanosine, adenine, guanine, hypoxanthine, xanthine, uric acid and pyrimidine bases were determined in the CSF of 18 children after simple febrile seizures and in a control group. There was no statistically significant difference between the two groups for any of these metabolites. This suggests that simple febrile seizures neither significantly disturb the metabolism of nucleotides, nucleosides or bases, nor significantly deplete neuron adenosine triphosphate ATP levels.

Cerebrospinal fluid calcium, parathyroid hormone, and monoamine and purine metabolites and the blood-brain barrier function in primary hyperparathyroidism.

Psychiatric disturbances are common in primary hyperparathyroidism (HPT), but their pathogenesis is essentially unknown. This study deals with cerebrospinal fluid (CSF) calcium homeostasis and its connection with parathyroid hormone (PTH), blood-brain barrier (BBB) function, and central monoamine and purine metabolites in patients with primary HPT. In 22 patients with primary HPT (serum calcium 2.85 +/- 0.21 mmol/l), the CSF concentrations of total and ionized calcium were higher (1.21 +/- 0.08 mmol/l, p less than 0.01, and 1.09 +/- 0.05 mmol/l, p less than 0.001, respectively) than in 11 normocalcemic reference subjects. The values correlated with serum calcium concentration (p less than 0.001) and CSF/serum albumin ratio, a measure of BBB permeability. The latter ratio was elevated in one-third of the patients with HPT, indicating BBB damage. CSF immunoreactive intact PTH was higher in the HPT patients than in the reference group (p less than 0.05), and serum and CSF PTH were positively correlated (p less than 0.05). The CSF levels of the monoamine metabolites 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) were lower, and the level of urate in CSF was higher, in the HPT patients than in the reference subjects, while there were no consistent differences in CSF hypoxanthine or xanthine. CSF 5HIAA correlated inversely with CSF ionized calcium (r = -0.42, p = 0.02). After parathyroid surgery, CSF calcium and urate decreased significantly and CSF monoamine metabolites increased slightly. The decrease in CSF ionized calcium correlated with the alleviation of psychiatric symptoms. The results indicate the importance of increased CSF calcium concentrations in patients with primary HPT and suggest a relation between central calcium regulation and central turnover of monoamines.

Opposite alterations in cerebrospinal fluid uridine after severe cerebral ischemia or intrathecal blood injection.

1. Rats which survived hypoglycemia by insulin, hypoxia by 10% O2, or ischemia by carotid ligation and hypotension to 40 mm Hg, evidenced no changes in cerebrospinal fluid (CSF) uridine. Animals which died soon after the above interventions or as a result of KCl-induced cardiac arrest had elevated CSF uridine concentrations. 2. Injection of whole blood or the soluble contents of lysed blood cells into the lateral ventricle of rats reduced CSF uridine to less than one-half normal at 24 hrs but values returned to normal 3 days later. Changes in hypoxanthine resembled those of uridine, but were less dramatic, whereas xanthine concentrations were largely unaltered. Intraventricular injection of plasma or saline did not alter CSF uridine. 3. It seems most likely that low CSF uridine concentrations previously reported in head injury patients may be secondary to the effects of blood cell contents in the cerebrospinal fluid, rather than responses to altered metabolism in neurons or glia cells.

[Metabolism of purine nucleotides in the central nervous system in patients with phosphoribosylpyrophosphate synthetase hyperactivity and neurosensory deafness]. / Metabolismo de nucleótidos purínicos en el sistema nervioso central de pacientes con sobreactividad de fosforribosilpirofosfato (PRPP) sintetasa y sordera neurosensorial.

The overactivity of PRPP synthetase is transmitted as a sex-linked abnormality, being characterized by uric acid overproduction and, in some patients, by muscular hypotonia, neurosensitive deafness and/or ataxia. The pathogenesis of these neurologic abnormalities is not yet known. The CSF concentrations of end products of the neuronal metabolism of purines--hypoxanthine for the adenine nucleotides and xanthine for guanine nucleotides--have not been previously studied in patients with overactivity of PRPP synthetase. We have evaluated the plasma and CSF levels of hypoxanthine and xanthine in a 8-year-old male with tophaceous gout and neurosensitive deafness and in his mother, who had gout without neurological involvement. PRPP synthetase overactivity was demonstrated in fibroblast culture; the male was hemizygote and his mother was heterozygotic. In 4 normal individuals, the plasma levels of hypoxanthine and xanthine were 1.7 +/- 0.4 microM and 0.9 +/- 0.2 microM (mean +/- SEM), respectively, while in in CSF they were 3.3 +/- 1.1 microM and 2.0 +/- 0.2 microM. The hemizygote male showed a considerable increase in hypoxanthine level (5.6 microM in plasma and 22.1 microM in CSF); the plasma and CSF xanthine levels were 1.8 and 4.5 microM, respectively. The heterozygotic female showed moderately increased plasma hypoxanthine levels (3.9 and 10.6 microM) and normal xanthine levels (1.3 and 1.8 microM). These results suggest an increase in the degradation of purine nucleotides in the central nervous system of patients with PRPP synthetase overactivity and neurological symptoms. The predominance of hypoxanthine over xanthine may indicate a greater increase of the degradation of adenine rather than guanine nucleotides.

Therapeutic criteria in hydrocephalic children.

The xanthine, hypoxanthine, and total oxypurine levels were determined in the CSF of 28 hydrocephalic patients (age from newborn to 2 years) and 8 healthy controls using HPLC. The Evans' index, the mean weekly increase in cranial circumference, and the intracranial pressure were also measured. Of the hydrocephalic patients 13 were self-compensated and the other 15 had a shunt implanted during the course of the study. The mean xanthine, hypoxanthine, and total oxypurine levels in the normal children were 5.20, 5.94, and 11.29 mumol/l, respectively. In the self-compensated hydrocephalics these levels were 5.17, 5.71, and 10.79 mumol/l, respectively. In the noncompensated hydrocephalics, they were 9.90, 9.91, and 19.82 mumol/l. The differences between the latter group and the first two are statistically significant (P less than 0.001). The mean Evans' index and the mean weakly increase in cranial circumference in the self-compensated hydrocephalics were 0.35 and 0.25 cm, respectively. In the noncompensated hydrocephalics, they were 0.55 and 0.95 cm. The differences between the two groups are statistically significant (P less than 0.001). Two weeks after implantation of shunts in the noncompensated cases, the mean xanthine, hypoxanthine, and total oxypurine levels fell to 4.22, 4.57, and 8.80 mumol/l, respectively. These changes are statistically significant (P less than 0.001). We think that the two criteria (clinical and biochemical) are equally useful for the prediction of self-compensation in hydrocephalic children and that the oxypurine values after shunt implantation can be used to monitor progress in noncompensated cases.

Cerebrospinal fluid nucleotide metabolites following short febrile convulsions.

Cerebrospinal fluid (CSF) markers of cerebral energy depletion were measured in 32 infants and children following short (less than 10 minutes) febrile convulsions, and in 19 controls. Specific and sensitive indices of high-energy phosphate compound depletion (hypoxanthine, xanthine and uridine) showed no marked changes. Values for patients and febrile controls were significantly higher than for afebrile controls, which is consistent with increased cerebral metabolism in febrile patients. There were no differences in pH, lactate or creatine kinase levels in the CSF of patients and controls. The results suggest that short febrile convulsions are benign and that in the absence of risk factors for the subsequent development of epilepsy, prophylactic anticonvulsant treatment is not indicated.