Mohs micrographic surgery: a technique for total margin assessment in veterinary cutaneous oncologic surgery.

Mohs micrographic surgery (MMS) is the gold standard for the excision of locally invasive cutaneous malignancies in human dermatological surgery. Using a unique horizontal sectioning technique, MMS enables 100% surgical margin assessment and provides the lowest recurrence rates for locally invasive tumours. The purposes of this preliminary study were to explore the feasibility of application of MMS in the veterinary setting and to establish practical advantages and limitations of its use in a pilot programme. It was hypothesized that MMS technique could provide 100% tumour margin assessment using frozen and/or formalin-fixed horizontal histopathologic sections. Tumour excision and colour-coded mapping were performed, and specimen tissue was fixed using either frozen sections or formalin-fixed sections. Horizontal sections were assessed for quality and presence and location of neoplastic cells based on the mapped orientation. The MMS technique was used in the excision of six squamous cell carcinomas and five mast cell tumours. In all cases, the MMS permitted 100% tumour margins examination.

Methyl analogues of the experimental Alzheimer drug phenserine: synthesis and structure/activity relationships for acetyl- and butyrylcholinesterase inhibitory action.

With the goal of developing potential Alzheimer's pharmacotherapeutics, we have synthesized a series of novel analogues of the potent anticholinesterases phenserine (2) and physostigmine (1). These derivatives contain methyl (3, 4, 6), dimethyl (5, 7, 8, 10, 11) and trimethyl (14) substituents in each position of the phenyl group of the phenylcarbamoyl moieties, and with N-methyl and 6-methyl substituents (12, 13, 31, 33). We also quantified the inhibitory action of these compounds against human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). An analysis of the structure/anticholinesterase activity relationship of the described compounds, together with molecular modeling, confirmed the catalytic triad mechanism of the binding of this class of carabamate analogues within AChE and BChE and defined structural requirements for their differential inhibition.

Insertion of an N-terminal 6-aminohexanoic acid after the 7 amino acid position of glucagon-like peptide-1 produces a long-acting hypoglycemic agent.

The use of glucagon-like peptide-1 (GLP-1) as a routine treatment for type 2 diabetes mellitus is undermined by its short biological half-life. A cause of degradation is its cleavage at the N-terminal HAE sequence by the enzyme dipeptidyl peptidase IV (DPP IV). To protect from DPP IV, we have studied the biological activity of a GLP-1 analog in which 6-aminohexanoic acid (Aha) is inserted between histidine and alanine at positions 7 and 8. We have compared the biological activity of this new compound, GLP-1 Aha(8), with the previously described GLP-1 8-glycine (GLP-1 Gly(8)) analog. GLP-1 Aha(8) (10 nM) was equipotent with GLP-1 (10 nM) in stimulating insulin secretion in RIN 1046-38 cells. As with GLP-1 Gly(8), the binding affinity of GLP-1 Aha(8) for the GLP-1 receptor in intact Chinese hamster ovary (CHO) cells expressing the human GLP-1 receptor (CHO/GLP-1R cells) was reduced (IC(50): GLP-1, 3.7 +/- 0.2 nM; GLP-1 Gly(8), 41 +/- 9 nM; GLP-1 Aha(8), 22 +/- 7 nM). GLP-1 Aha(8) was also shown to stimulate intracellular cAMP production 4-fold above basal at concentrations as low as 0.5 nM. However, it exhibited a higher ED(50) when compared to GLP-1 and GLP-1 Gly(8) (ED(50): GLP-1, 0.036 +/- 0.002 nM, GLP-1 Gly(8), 0.13 +/- 0.02 nM, GLP-1 Aha(8), 0.58 +/- 0.03 nM). A series of D-amino acid-substituted GLP-1 compounds were also examined to assess the importance of putative peptidase-sensitive cleavage sites present in the GLP-1 molecule. They had poor binding affinity for the GLP-1 receptor, and none of these compounds stimulated the production of intracellular cAMP in CHO/GLP-1R cells or insulin secretion in RIN 1046-38 cells. GLP-1 Aha(8) (24 nmol/kg) administered sc to fasted Zucker (fa/fa) rats (mean blood glucose, 195 +/- 32 mg/dl) lowered blood glucose levels to a nadir of 109 +/- 3 mg/dl, and it remained significantly lower for 8 h. Matrix-assisted linear desorption ionization-time of flight mass spectrometry of GLP-1 Aha(8) incubated with DPP IV (37 C, 2 h) did not exhibit an N-terminal degradation product. Taken together, these results show that insertion of Aha after the 7 position in GLP-1 produces an effective, long-acting GLP-1 analog, which may be useful in the treatment of type 2 diabetes mellitus.

A new therapeutic target in Alzheimer's disease treatment: attention to butyrylcholinesterase.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the elderly, characterised by widespread loss of central cholinergic function. The only symptomatic treatment proven effective to date is the use of cholinesterase (ChE) inhibitors to augment surviving cholinergic activity. ChE inhibitors act on the enzymes that hydrolyse acetylcholine (ACh) following synaptic release. In the healthy brain, acetylcholinesterase (AChE) predominates (80%) and butyrylcholinesterase (BuChE) is considered to play a minor role in regulating brain ACh levels. In the AD brain, BuChE activity rises while AChE activity remains unchanged or declines. Therefore both enzymes are likely to have involvement in regulating ACh levels and represent legitimate therapeutic targets to ameliorate the cholinergic deficit. The two enzymes differ in location, substrate specificity and kinetics. Recent evidence suggests that BuChE may also have a role in the aetiology and progression of AD beyond regulation of synaptic ACh levels. Experimental evidence from the use of agents with enhanced selectivity for BuChE (cymserine, MF-8622) and ChE inhibitors such as rivastigmine, which have a dual inhibitory action on both AChE and BuChE, indicate potential therapeutic benefits of inhibiting both AChE and BuChE in AD and related dementias. The development of specific BuChE inhibitors and the continued use of ChE inhibitors with the ability to inhibit BuChE in addition to AChE should lead to improved clinical outcomes.

Time course of pharmacodynamic and pharmacokinetic effects of physostigmine assessed by functional brain imaging in humans.

In imaging studies of brain functions using pharmacological probes, identification of the time point at which central effects of intravenously infused drugs become stable is crucial to separate the effects of experimental variables from the concomitant changes in drug effects over time. We evaluated the time courses of the pharmacokinetics and pharmacodynamics, including butyrylcholinesterase inhibition and central neural responses, of physostigmine in healthy young subjects. Ten positron emission tomography (PET) scans that alternated between a rest condition (eyes open, ears unplugged) and a working memory for faces (WM) task were acquired in healthy subjects. Subjects in the drug group received a saline infusion for the first two scans, providing a baseline measure, then received an infusion of physostigmine for all subsequent scans. Subjects in the control group received a placebo infusion of saline for all scans. Physostigmine plasma levels and percent butyrylcholinesterase inhibition increased over time (p < 0. 0001), and both became stable by 40 min. Physostigmine decreased reaction time (RT) (p = 0.0005), and this effect was detected after 20 min of infusion and stable thereafter. Physostigmine also decreased regional cerebral blood flow (rCBF) in right prefrontal cortex during task (p = 0.0002), and this effect was detected after 40 min of infusion and stable thereafter. No change in RT or rCBF was observed in the control group. These results indicate that a 40-min infusion of physostigmine was necessary to obtain stable central effects. More generally, we have demonstrated that experimental effects can vary with time, especially during the initial phases of a drug infusion, indicating that it is critical that these changes are controlled.

Exendin-4 decelerates food intake, weight gain, and fat deposition in Zucker rats.

Exendin-4 is a 39 amino acid peptide produced in the salivary gland of the Gila monster lizard. It has a 53% amino acid homology to the incretin hormone glucagon-like peptide-1 (GLP-1). Exendin-4 induces insulin release through activation of the GLP- 1 receptor but is a much more potent insulinotropic agent than GLP-1. Of critical importance for its potential use as a treatment for diabetes is its much longer biological effect in vivo. Previous studies involving once daily administration of exendin-4 over 13 weeks to db/db mice demonstrated that it lowers hemoglobin A1c (HbA1c), a marker of mean blood glucose levels. Food consumption in the treated animals dropped over the first 4 days and then increased to a level comparable with that of the untreated animals. In this study, we initially examined the effect of once daily injections (over 14 days) on the food consumption of Zucker fatty rats. We observed an immediate reduction in food intake which then leveled off(after 5 days) to match that of the untreated animals. Subsequently we injected the same animals twice daily (treatment period of 56 days in total) and observed a sustained reduction in food intake and weight-gain. This was matched by a reduction in the critical parameters of HbA1c, fasting blood glucose and plasma insulin. MRI imaging of the abdominal regions of the animals showed that initially only the amount of fat deposited in the sc region was reduced after 4 weeks exendin-4 treatment. At the 8-week time point there was a corresponding decrease in the amount of visceral fat deposition. The combination of appetite reduction, decreased fat deposition and an improvement in the parameters associated with glucose intolerance makes a case for the use of exendin-4 as a treatment for diabetes.

The experimental Alzheimer drug phenserine: preclinical pharmacokinetics and pharmacodynamics.

Phenserine, a phenylcarbamate of physostigmine, is a new potent and highly selective acetylcholinesterase (AChE) inhibitor, with a > 50-fold activity versus butyrylcholinesterase (BChE), in clinical trials for the treatment of Alzheimer's disease (AD). Compared to physostigmine and tacrine, it is less toxic and robustly enhances cognition in animal models. To determine the time-dependent effects of phenserine on cholinergic function, AChE activity, brain and plasma drug levels and brain extracellular acetylcholine (ACh) concentrations were measured in rats before and after phenserine administration. Additionally, its maximum tolerated dose, compared to physostigmine and tacrine, was determined. Following i.v. dosing, brain drug levels were 10-fold higher than those achieved in plasma, peaked within 5 min and rapidly declined with half-lives of 8.5 and 12.6 min, respectively. In contrast, a high (> 70%) and long-lasting inhibition of AChE was achieved (half-life > 8.25 h). A comparison between the time-dependent plasma AChE inhibition achieved after similar oral and i.v. doses provided an estimate of oral bioavailability of 100%. Striatal, in vivo microdialysis in conscious, freely-moving phenserine-treated rats demonstrated > 3-fold rise in brain ACh levels. Phenserine thus is rapidly absorbed and cleared from the body, but produces a long-lasting stimulation of brain cholinergic function at well tolerated doses and hence has superior properties as a drug candidate for AD. It selectively inhibits AChE, minimizing potential BChE side effects. Its long duration of action, coupled with its short pharmacokinetic half-life, reduces dosing frequency, decreases body drug exposure and minimizes the dependence of drug action on the individual variations of drug metabolism commonly found in the elderly.

Synthesis of novel phenserine-based-selective inhibitors of butyrylcholinesterase for Alzheimer's disease.

Four novel analogues (8-11) of cymserine (2) were synthesized by methods similar to those recently developed for the total syntheses of N8-norphenserine (Yu, Q. S.; et al. J. Med. Chem. 1997, 40, 2895-2901) and N1,N8-bisnorphenserine (Yu, Q. S.; et al. J. Med. Chem. 1998, 41, 2371-2379). As our structure-activity studies predicted, these compounds are highly potent and selective inhibitors of human butyrylcholinesterase (BChE) and will test the novel hypothesis that BChE inhibitors are useful in the treatment of Alzheimer's disease. In a similar manner, the same modifications that provided BChE selectivity were applied to the acetylcholinesterase (AChE)-selective inhibitor, tolserine (5), to provide the novel tolserine analogues 12-15. As predicted, these modifications altered the AChE-selective action of tolserine (5) to favor a lack of cholinesterase enzyme subtype selectivity.

Once daily injection of exendin-4 to diabetic mice achieves long-term beneficial effects on blood glucose concentrations.

Glucagon-like peptide-1 is the main hormonal mediator of the enteroinsular axis. Recently, it has additionally received considerable attention as a possible new treatment for Type II (non-insulin-dependent) diabetes mellitus. Its major disadvantage is that its duration of action is too short to achieve good 24-h metabolic control. Exendin-4, which is produced in the salivary glands of Gila monster lizards, is structurally similar to glucagon-like peptide-1 and shares several useful biological properties with glucagon-like peptide-1. It binds the glucagon-like peptide-1 receptor, stimulates insulin release and increases the cAMP production in beta cells. We report that exendin-4 is a more potent insulinotropic agent when given intravenously to rats than is glucagon-like peptide-1 (ED50 0.19 nmol/kg for glucagon-like peptide-1 vs 0.0143 nmol/kg for exendin-4) and causes a greater elevation in cAMP concentrations in isolated islets. Of even greater interest we found that when given intraperitoneally only once daily to diabetic mice it had a prolonged effect of lowering blood glucose. After 1 week of treatment blood glucoses were 5.0+/-2.6 mmol/l compared to diabetic concentrations of 13.2+/-2.8 mmol/l. After 13 weeks of daily treatment HbA1c was 8.8+/-0.4% in non-treated diabetic animals compared with 4.7+/-0.25% in treated diabetic animals. Blood glucoses also were lower (p < 0.005) and insulin concentrations higher (p < 0.02) in the treated animals. Exendin-4 could therefore be preferable to glucagon-like peptide-1 as a long-term treatment of Type II diabetes.

Syntheses and anticholinesterase activities of (3aS)-N1, N8-bisnorphenserine, (3aS)-N1,N8-bisnorphysostigmine, their antipodal isomers, and other potential metabolites of phenserine.

Hydrolysis of the carbamate side chains in phenserine [(-)1] and physostigmine [(-)2] yields the metabolite (-)-eseroline (3), and the red dye rubreserine (4) on air oxidation of the former compound. Both compounds lacked anticholinesterase activity in concentrations up to 30 mM, which would be unachievable in vivo. A second group of potential metabolites of 1 and 2 are the N1,N8-bisnorcarbamates (-)9 and (-)10, prepared from (3aS)-N8-benzylnoresermethole (-)12 by the carbinolamine route. These entirely novel compounds proved to be highly potent inhibitors of acetylcholinesterase [(-)9] and of acetyl- and butyrylcholinesterase (AChE and BChE) [(-)10], respectively. To elucidate further the structure/anticholinesterase activity relationship of the described compounds, the antipodal isomers (3aR)-N1,N8-bisnorcarbamates (+)9 and (+)10 were likewise synthesized from (3aR)-N8-benzylnoresermethole (+)12 and assessed. The compounds possessed moderate but less potent anticholinesterase activity, with the same selectivity as their 3aS enantiomers. Finally, the anticholinesterase activities of intermediates N1, N8-bisnorbenzylcarbamates (-)18, (-) 19, (+)18, and (+)19, also novel compounds, were additionally measured. The 3aS enantiomers proved to be potent and selective inhibitors of BChE, particularly (-)19, whereas the antipodal isomers lacked activity.

The effects of GLP-1 on insulin release in young and old rats in the fasting state and during an intravenous glucose tolerance test.

Glucose intolerance is a common feature of the aging process, and aging per se is an etiologic factor for Type II diabetes mellitus. To characterize the beta cell abnormalities that occur with aging, we looked at the serum glucose and insulin levels of six young (3-month) and six old (22-month) Wistar rats at 0, 2, 4, 7, 10, 15, 20, and 30 minutes after an intravenous glucose load (IVGTT, 0.5 g/kg glucose). We found that the fasting glucose and insulin levels were not significantly different between young and old rats. However, peak glucose levels were significantly higher in the old (349 +/- 10 mg/dl) compared to the young (250 +/- 7 mg/dl) animals (p < .0001). Insulin levels in the young animals peaked at 2 minutes (859 +/- 171 pmol/l) with a quick return toward fasting levels by 7 minutes. The old animals had a delayed and blunted insulin response to glucose, achieving lower peak insulin levels (656 +/- 164 pmol/l) 7 minutes after the glucose load. As insulin levels are also positively modulated by incretin hormones, we quantitated the fasting insulin responses of young and old animals to .05, 0.1, 0.2, 0.4, and 0.5 nmol/kg intravenous glucagon-like peptide-1 (GLP-1), the most potent incretin known. Insulin responses were similar in both age groups, with maximum insulin responses seen at 0.4 nmol/kg. GLP-1, in conjunction with the IVGTT, restored the acute insulin response to glucose and increased the clearance of glucose in the old animals. It therefore appears that old animals have an impaired glucose-mediated insulin release but maintain their insulin responsivity to GLP-1. This makes it a likely candidate in the treatment of Type II diabetes.

Total syntheses and anticholinesterase activities of (3aS)-N(8)-norphysostigmine, (3aS)-N(8)-norphenserine, their antipodal isomers, and other N(8)-substituted analogues.

N(8)-Benzylesermethole (6) was prepared from 5-methoxytryptamine (1) in five steps. Resolution of compound 6 by dibenzoyl- and ditoluyltartaric acid provided enantiomers (-)- and (+)-7. After demethylation, reaction with isocyanates and catalytic debenzylation over hydrogen, the total syntheses of (-)- and (+)-N(8)-norphysostigmine [(-)- and (+)-11] and (-)- and (+)-N(8)-norphenserine [(-)- and (+)-12] were accomplished, (-)-N(8)-Norphysostigmine [(-)-11] and (-)-N(8)-norphenserine [(-)-12] were also obtained by transformations of natural physostigmine [(-)-13] and phenserine [(-)-14] prepared from (-)-13. The absolute configurations and optical purity of compounds (-)-11, (-)-12, (+)-11, and (+)-12 were confirmed by a comparison of their optical rotations with those of the compounds synthesized from physostigmine [(-)-13]. The anticholinesterase activities of N(8)-nor- and N(8)-substituted analogues, (-)- and (+)-9, -10, -11, -12, 15, and 16, were compared with those of physostigmine [(-)- and (+)-13] and phenserine [(-)- and (+)-14] and are reported.

Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats.

Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg-1.min-1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04+/-0.92 mmol/liter, P < 0.01) than saline-treated animals (11.61+/-0.23 mmol/liter). At 30 min the plasma glucose was still lower in the GLP-1-treated animals (8.61+/-0.39 mmol/liter, P < 0.05) than saline-treated animals (10.36+/-0.43 mmol/liter). This decrease in glucose levels was reflected in the higher insulin levels attained in the GLP-1-treated animals (936+/-163 pmol/liter vs. 395+/-51 pmol/liter, GLP-1 vs. saline, respectively, P < 0.01), detected 15 min after glucose injection. GLP-1 treatment also increased pancreatic insulin, GLUT2, and glucokinase mRNA in the old rats. The effects of GLP-1 were abolished by simultaneous infusion of exendin [9-39], a specific antagonist of GLP-1. GLP-1 is therefore able to reverse some of the known defects that arise in the beta cell of the pancreas of Wistar rats, not only by increasing insulin secretion but also by inducing significant changes at the molecular level.

Application of stable isotope tracer combined with mass spectrometric detection for studying myo-inositol uptake by cultured neurons from fetal mouse: effect of trisomy 16.

A gas chromatographic (GC)/mass spectrometric method for studying myo-inositol uptake by neurons in vitro is described. Cultured cortical neurons from fetuses of diploid and trisomy 16 mouse (animal model for Down syndrome) were incubated with a physiological concentration of hexadeuterated myo-inositol for 2-40 min. Washed cells were lysed and scyllo-inositol (internal standard) was added to the intracellular material which contained labeled myo-inositol taken up by the cells as well as the endogenous, unlabeled myo-inositol. The samples were evaporated to dryness and the analytes were converted into acetate derivatives. The components were separated by capillary GC, and the m/z 379 ion for labeled myo-inositol and the m/z 373 ion for myo-inositol and scyllo-inositol generated by chemical ionization in an ion trap mass spectrometer were monitored. Quantitation of the deuterium-labeled myo-inositol taken up by the neuron along with endogenous myo-inositol was achieved for 2-40 min of incubation. The labeled myo-inositol uptake was linear for up to 20 min and was Na+ dependent in these neurons. This non-radioisotope method was used to demonstrate a significant (40%) increase in the rate of myo-inositol uptake by cortical neurons from the trisomy 16 mouse relative to control neurons. An increased myo-inositol uptake is consistent with evidence that the myo-inositol transporter gene is on both human chromosome 21 and mouse chromosome 16, and that myo-inositol concentrations are elevated in cerebrospinal fluid from adult Down syndrome individuals and brains from the fetal trisomy 16 mouse.

Clinical pharmacokinetics of arecoline in subjects with Alzheimer's disease.

OBJECTIVE: To study the pharmacokinetics and pharmacodynamics of intravenously administered arecoline in subjects with Alzheimer's disease. METHODS: Plasma arecoline concentrations were measured during and after high-dose (i.e., 5 mg intravenously over 30 minutes) and up to 2 weeks of continuous multiple-dose steady-state intravenous infusions of arecoline in 15 subjects with mild to moderate Alzheimer's disease. During multiple-dose infusions, the dose of arecoline was escalated from 0.5 to 40 mg/day. Psychometric tests were administered at baseline and every other dose to determine an "optimal dose" for each subject. This dose then was administered for 1 week using a randomized, placebo-controlled, double blind, crossover design. Plasma drug concentrations were measured by GC-MS. RESULTS: The optimal dose of arecoline varied fourfold across subjects (4 mg/day, n = 6; 16 mg/day, n = 3) with mean plasma half-lives of 0.95 +/- 0.54 and 9.3 +/- 4.5 (SD) minutes. Clearance and volume of distribution were 13.6 +/- 5.8 L/min and 205 +/- 170 (SD) L, respectively. At the dose that optimized memory, the mean plasma level was 0.31 +/- 0.14 (SD) ng/ml, and it predicted the optimal dose in all subjects. CONCLUSIONS: Because optimal dose variation is due to differing plasma kinetics, the plasma arecoline level measured at a single infusion rate can be used to choose the optimal dose for memory enhancement in patients with Alzheimer's disease.

Cerebrospinal fluid and plasma distribution of myo-inositol and other polyols in Alzheimer disease.

Previous studies suggest the presence of increased concentrations of cerebral myo-inositol in Alzheimer disease (AD). To characterize this abnormality further, we quantified myo-inositol and several other polyols in cerebrospinal fluid (CSF) and plasma from 10 AD subjects and 10 healthy age-matched controls by using a gas chromatographic-mass spectrometric technique. The mean CSF concentration and CSF/plasma concentration ratio of myo-inositol in AD were not significantly different from those determined in control subjects. Also, concentration profiles of other polyols were not significantly altered in AD. CSF and plasma myo-inositol concentrations were correlated in control subjects but not in AD subjects. However, a significant correlation between CSF and plasma 1,5-anhydrosorbitol (a polyol internal control) concentrations observed in control subjects was retained in AD subjects.

Brain accumulation of myo-inositol in the trisomy 16 mouse, an animal model of Down's syndrome.

myo-Inositol and several other polyols were measured in the tissues of the trisomy 16 mouse (animal model of Down's Syndrome; human trisomy 21) and diploid controls. myo-Inositol was found to be selectively elevated in the brain of the trisomy 16 mouse. However, peripheral tissues showed no elevation. These results are consistent with the cerebrospinal fluid and plasma data reported previously on myo-inositol in Down's Syndrome subjects.

Polyol profiles in Down syndrome. myo-Inositol, specifically, is elevated in the cerebrospinal fluid.

Polyols are reduction products of aldoses and ketoses; their concentrations in tissues can reflect carbohydrate metabolism. Several polyol species were quantitated in cerebrospinal fluid (CSF) and plasma from 10 Down Syndrome (trisomy 21) subjects between the ages of 22 and 63 years (3 of whom were demented) and from 10 healthy age-matched controls, using a gas chromatographic/mass spectrometric technique. The mean CSF concentration and the mean CSF/plasma concentration ratio of myo-inositol were significantly elevated in Down syndrome compared with controls, but were not correlated with the presence of dementia in the Down subjects. Plasma myo-inositol was not significantly altered in these subjects. No significant difference between Down syndrome and controls was found for CSF concentrations of mannitol, sorbitol, galactitol, ribitol, arabitol, or 1,5-anhydrosorbitol, but plasma mannitol, ribitol and arabitol were elevated in Down syndrome. The present observation provides new impetus for studying synthesis and transport of myo-inositol as well as phosphatidylinositol cycle in trisomy 21 disorder.

Capillary gas chromatography combined with ion trap detection for quantitative profiling of polyols in cerebrospinal fluid and plasma.

Polyol species in cerebrospinal fluid and plasma--ribitol, arabitol, xylitol, 1,5-anhydrosorbitol, myo-inositol, mannitol, sorbitol, and galactitol--simultaneously were quantitated by a capillary gas chromatography/ion trap (mass spectrometric) detection method. The details of the methodology are discussed and the results of analysis of polyols in healthy human subjects are reported. Microliter volumes of cerebrospinal fluid or plasma were mixed with internal standard (deuterium labeled myo-inositol), deproteinized, and evaporated to dryness. Polyols were acetylated in the presence of pyridine catalyst and washed with sodium bicarbonate solution and the acetate derivatives were recovered. Standard curve solutions were similarly treated. The polyol components were resolved on a capillary column bonded with 50% phenyl-50% methyl polysiloxane. Chemical ionization mass spectra for the acetate derivatives of polyols were generated in an ion trap using acetonitrile as reagent gas. Each polyol yielded a fragment ion in 100% abundance arising probably from the loss of one acetate moiety from the protonated molecule. These ions were monitored. The relative standard deviation (within-day) for quantitation of polyols was not greater than 8% for cerebrospinal fluid and 15% for plasma matrix. A polyol profile in cerebrospinal fluid and plasma was determined in healthy human subjects and a cerebrospinal fluid/plasma concentration ratio larger than 1.0 was found for all polyol species except 1,5-anhydrosorbitol and xylitol. This assay technique will be used to study the role of polyols in central nervous system diseases.

Assay of myo-inositol in cerebrospinal fluid and plasma by chemical ionization mass spectrometry of the hexaacetate derivative.

The paper describes a capillary gas chromatographic/mass spectrometric technique to quantitate myo-inositol in cerebrospinal fluid (CSF) and plasma. A highly abundant fragment ion, m/z 373, for the hexaacetate derivative of myo-inositol was generated by chemical ionization (CI). This ion and the analogous ion of hexadeuterated myo-inositol (the internal standard), m/z 379, were both monitored in the assay. CI was performed with acetonitrile vapor in an ion trap mass spectrometer. Microliter quantities of CSF or plasma were mixed with the internal standard, dried and acetylated. After a post-derivatization clean-up, samples were analyzed on a capillary gas chromatograph interfaced with a Finnigan ion trap. Standard curves constructed for both CSF and plasma were linear and reproducible. Absence of matrix effects and good precision in the results indicate the suitability of the technique for critical assays. Results from the determination of myo-inositol in the CSF and plasma of healthy subjects and those with neurological deficits are reported.