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PubMed2002

Vacancy ion-exclusion chromatography of carboxylic acids on a weakly acidic cation-exchange resin.

In this preliminary study, a new approach to ion-exclusion chromatography is proposed to overcome the relatively poor conductivity detection response which occurs in ion-exclusion chromatography when acids are added to the eluent in order to improve peak shape. This approach, termed vacancy ion-exclusion chromatography, requires the sample to be used as eluent and a sample of water to be injected onto a weakly acidic cation-exchange column (TSKgel OApak-A). Vacancy peaks for each of the analytes appear at the retention times of these analytes. Highly sensitive conductivity detection is possible and sharp, well-shaped peaks are produced, leading to efficient separations. Retention times were found to be affected by the concentration of the analytes in the eluent, and also by the presence of an organic modifier such as methanol in the eluent. Detection limits for oxalic, formic, acetic, propionic, butyric and valeric acids were 0.1, 0.2, 0.3, 0.3, 0.4 and 0.5 microM, respectively, and linear ranges for some acids extended over two orders of magnitude. Precision values for retention times were 0.21% and for peak areas were <1.90%. The vacancy ion-exclusion chromatography method was found to give detection responses four to 10 times higher than conventional ion-exclusion chromatography using sulfuric acid eluent and two to five times higher than conventional ion-exclusion chromatography using benzoic acid eluent.

Cation Exchange Resins↗
PubMed1994

Role of guanylyl cyclase and cGMP-dependent protein kinase in long-term potentiation.

Several lines of evidence suggest that cyclic GMP might be involved in long-term potentiation (LTP) in the hippocampus. Arachidonic acid, nitric oxide and carbon monoxide, three molecules that have been proposed to act as retrograde messengers in LTP, all activate soluble guanylyl cyclase. We report here that an inhibitor of guanylyl cyclase blocks the induction of LTP in the CA1 region of hippocampal slices. Conversely, cGMP analogues produce long-lasting enhancement of the excitatory postsynaptic potential if they are applied at the same time as weak tetanic stimulation of the presynaptic fibres. The enhancement is spatially restricted, is not blocked by valeric acid (APV), nifedipine, or picrotoxin, and partially occludes LTP. This synaptic enhancement may be mediated by the cGMP-dependent protein kinase (PKG). Inhibitors of PKG block the induction of LTP, and activators of PKG produce activity-dependent long-lasting enhancement. These results suggest that guanylyl cyclase and PKG contribute to LTP, possibly as activity-dependent presynaptic effectors of retrograde messengers.

Action Potentials↗
PubMed1986

Stimulation of receptors of gamma-aminobutyric acid modulates the release of cholecystokinin-like immunoreactivity from slices of rat neostriatum.

Slices of rat dorsal neostriatum were incubated in Krebs-Henseleit medium and the release of cholecystokinin-like immunoreactivity (CCK-IR) was induced by veratridine or high concentrations of K+. It was investigated whether drugs which act at receptors for gamma-aminobutyric acid (GABA) affected the release. The GABAA-receptor agonists muscimol and isoguvacine enhanced the veratridine-induced release of CCK-IR. This effect was abolished by the GABAA-receptor antagonist, bicuculline. When used alone, bicuculline decreased the release. The GABAB-receptor agonist, (-)-baclofen, decreased the veratridine-induced release of CCK-IR. The stereoisomer (+)-baclofen, which has low intrinsic activity, had no effect when used alone, but antagonized the effect of (-)-baclofen as did delta-amino-n-valeric acid, another antagonist at GABAB-receptors. When the release of CCK-IR was stimulated by K+ (40 mM) in the presence of tetrodotoxin, it was no longer affected by GABAA-receptor agonists or antagonists. Thus, their sites of action were probably not in the immediate vicinity of the nerve-endings which release CCK-IR. Under these conditions, stimulation of GABAB-receptors still reduced the release of CCK-IR. Therefore, it is concluded that these receptors are in the immediate vicinity of or even on the terminals which release CCK-IR.

Animals↗
PubMed2000

Dietary carbohydrates affect caecal fermentation and modify nitrogen excretion patterns in rats. I. Studies with protein-free diets.

In a two-factorial experiment on 96 young male rats, the effects of substituting 10% raw potato starch (PS), pectins (PEC), or cellulose (CEL) for corn starch (CS) were studied using an unsupplemented protein-free (PF) diet or a PF diet supplemented either with DL-methionine or urea. The pH and the short chain fatty acids (SCFA) content in caecal digesta, as well as caecal digesta and tissue weights were determined and used as the criteria of caecal fermentation intensity. Blood urea level, amount of N excreted via faeces and urine, DAPA content, and amino acid composition of faecal protein were analyzed as indices of protein metabolism. A 10-day adaptation period to the carbohydrates fed with the casein diet preceded the experimental period of feeding the respective carbohydrates with protein-free diets. Dietary carbohydrates significantly influenced total and individual SCFA content in caecal digesta, as well as other parameters related to the intensity of fermentation. Potato starch and pectins were more intensively fermented than cellulose. Supplementation of the PF diet with methionine and urea affected only caecal isobutyric and valeric acid content in a way dependent on the carbohydrates present in the diet. Carbohydrates significantly altered the routes of N excretion. Faecal excretion was increased by all carbohydrates studied compared to corn starch, pectins had the most marked effect. Urinary excretion was significantly increased by cellulose (as compared with the PEC and PS groups) and decreased by pectins as compared with all other groups. There was an interaction between the effects of carbohydrates and type of protein-free diet on faecal and urinary excretion. The sum of amino acids in faecal protein was the lowest on the PEC diet, but the amino acid composition expressed as a per cent of total amino acid content was similar in all groups. It can be concluded that dietary carbohydrates alter the excretion patterns of endogenous nitrogen in rats in different ways and that this effect is related to the intensity of their fermentation in the hind gut.

Animals↗
PubMed2006

Improved bioproduction of short-chain fatty acids (SCFAs) from excess sludge under alkaline conditions.

The production of short-chain fatty acids (SCFAs) from excess sludge was conducted in batch fermentation tests at different pH values ranging from 4.0 to 11.0. Experimental results of the impacts of different pHs on SCFAs production showed that during the first 8-day fermentation time the total SCFAs production at either pH 9.0 or pH 10.0 was much greater than that at acidic or neutral pH, and the maximal yield of 256.2 mg SCFAs-COD per gram of volatile suspended solids (VSS) was at pH 10.0, which was, respectively, over3 and 4times that at pH 5.0 and uncontrolled pH. Clearly, SCFAs production from excess sludge could be significantly improved and maintained stable by controlling the fermentation pH at 10.0. The composition of SCFAs and the percent distribution of individual SCFAs accounting for total SCFAs at pH 10.0 were analyzed. The SCFAs consisted of acetic, propionic, iso-butyric, n-butyric, iso-valeric, and n-valeric acids, and acetic acid was the most prevalent product with a fraction of 40-55%. Because the results of this study were differentfrom those of previous studies of SCFAs production, the mechanism of increased SCFAs production under alkaline conditions was investigated. Results showed that as soluble COD increased, more soluble protein was provided as the substrate for producing SCFAs. In addition, less or even no SCFAs were consumed by methanogens at alkaline pH, so the SCFAs production was therefore remarkably improved. Further investigation revealed thatthe formation of SCFA at pH 10.0 was dominated by biological effects rather than by chemical hydrolysis.

Biodegradation, Environmental↗
PubMed1997

Influence of dietary protein supplements on the formation of bacterial metabolites in the colon.

BACKGROUND: To evaluate the influence of increased dietary protein intake on bacterial colonic metabolism in healthy volunteers. METHODS: Short chain fatty acids, ammonia, and volatile organic compounds in faecal samples, and phenols in the urine of five volunteers were measured after one week of basal nutrient intake and and after one week of a diet supplemented with a protein rich food (Fortimel; Nutricia, Zoetermeer, The Netherlands). Paired t tests and factor analysis were used for statistical analysis. RESULTS: Total energy and resistant carbohydrate intake remained unchanged in each study period. The percentage energy intake delivered as dietary protein, increased significantly (from 15.4% to 23.8%; p = 0.007) during supplement intake. A significant increase in faecal ammonia (p = 0.002), faecal valeric acid (p = 0.02), and urinary p-cresol (p = 0.04) was noted during supplementary protein intake. A total of 120 different volatile compounds were isolated from the faecal samples of which 10 increased significantly during dietary protein supplementation. The change in volatile pattern, especially for S containing metabolites, was clearly shown by a factor analysis model which made a distinction between the two dietary regimens for all volunteers. CONCLUSION: An increase in dietary protein leads to altered products formation by colonic metabolism, mainly reflected by an increase in faecal ammonia, faecal volatile S substances, and urinary p-cresol.

Adult↗
PubMed1968

Sex attractant of sugar beet wireworm: identification and biological activity.

The sex attractant produced by adult females of the sugar beet wire-worm, Limonius californicus (Mannerheim) has been isolated and identified as valeric acid. In the laboratory, male wireworm beetles are repelled by the pure attractant but are drawn with intense sexual excitement to its dilute solutions; in the field, male beetles are lured from a distance of 12 meters. The pheromone occurs in unusually large amounts in the female's body.

Animals↗
PubMed1969

Biochemical decomposition of the herbicide N-(3,4-dichlorophenyl)-2-methylpentanamide and related compounds.

Organisms capable of decomposing N-(3,4-dichlorophenyl)-2-methylpentanamide (Karsil) were isolated, identified, and tested for their ability to hydrolyze this herbicide. Primary products of Karsil decomposition by cells and cell-free extracts of a Penicillium sp. were identified as 2-methyl-valeric acid and 3,4-dichloroaniline. The Karsil acylamidase (EC 3.5.1.a aryl acylamine amidohydrolase) was an induced enzyme. It was partially purified and tested for its ability to hydrolyze 25 related compounds. Some relations between the structures of these compounds and their susceptibility to enzymatic hydrolysis were discerned.

Amidohydrolases↗
PubMed2003

Optimizing the level of wet corn gluten feed in the diet of lactating dairy cows.

Twenty-four multiparous Holstein cows averaging 566 +/- 43 kg of body weight and 83 +/- 49 d in lactation were assigned to treatments stratified by age, days in milk, and milk yield to evaluate the effects of feeding increasing levels of wet corn gluten feed (WCGF) on lactational performance and milk composition. Complete diets containing 0, 15, 30, or 45% of the total ration dry matter (DM) as WCGF were formulated to be 17.2% crude protein and 1.72 Mcal of NE(L) per kilogram of DM, and fed twice daily to individual cows in Calan gates for 15 wk. All diets had a positive metabolizable protein balance. WCGF did not alter DM intake, but feed intake variance tended to be more consistent among cows fed 15 and 30% WCGF (DM basis). Weight gain was numerically greatest for those cows receiving 45% WCGF. Efficiency of energy and protein utilization was not different among treatments. Milk components of fat, protein, and casein were not different among treatments. Milk urea nitrogen was greater for cows on WCGF. Serum urea nitrogen was greatest in cows fed diets containing 15 and 45% WCGF. Serum insulin was lowest in the groups receiving 30 and 45% WCGF, but serum glucose and total protein were unaffected. The concentration of the ruminal volatile fatty acid, valerate, was greater in cows on the WCGF diet and highest in cows fed 30% WCGF. Ruminal ammonia was greatest in cows receiving 30% WCGF. It was estimated that 18.6% of the dietary DM fed as WCGF as a replacement for both portions of the concentrate and the forage in similar diets would have maximized milk yield without negatively affecting milk composition or feed efficiency.

Ammonia↗
PubMed2006

Decomposition of 1,4-dioxane by advanced oxidation and biochemical process.

This study was undertaken to determine the optimal decomposition conditions when 1,4-dioxane was degraded using either the AOPs (Advanced Oxidation Processes) or the BAC-TERRA microbial complex. The advanced oxidation was operated with H2O2, in the range 4.7 to 51 mM, under 254 nm (25 W lamp) illumination, while varying the reaction parameters, such as the air flow rate and reaction time. The greatest oxidation rate (96%) of 1,4-dioxane was achieved with H2O2 concentration of 17 mM after a 2-hr reaction. As a result of this reaction, organic acid intermediates were formed, such as acetic, propionic and butyric acids. Furthermore, the study revealed that suspended particles, i.e., bio-flocs, kaolin and pozzolan, in the reaction were able to have an impact on the extent of 1,4-dioxane decomposition. The decomposition of 1,4-dioxane in the presence of bio-flocs was significantly declined due to hindered UV penetration through the solution as a result of the consistent dispersion of bio-particles. In contrast, dosing with pozzolan decomposed up to 98.8% of the 1,4-dioxane after 2 hr of reaction. Two actual wastewaters, from polyester manufacturing, containing 1,4-dioxane in the range 370 to 450 mg/L were able to be oxidized by as high as 100% within 15 min with the introduction of 100:200 (mg/L) Fe(II):H202 under UV illumination. Aerobic biological decomposition, employing BAC-TERRA, was able to remove up to 90% of 1,4-dioxane after 15 days of incubation. In the meantime, the by-products (i.e., acetic, propionic and valeric acid) generated were similar to those formed during the AOPs investigation. According to kinetic studies, both photo-decomposition and biodegradation of 1,4-dioxane followed pseudo first-order reaction kinetics, with k = 5 x 10(-4) s(-1) and 2.38 x 10(-6) s(-1), respectively. It was concluded that 1,4-dioxane could be readily degraded by both AOPs and BAC-TERRA, and that the actual polyester wastewater containing 1,4-dioxane could be successfully decomposed under the conditions of photo-Fenton oxidation.

Biochemical Phenomena↗
PubMed1992

Receptor subtypes involved in callosally-induced postsynaptic potentials in rat frontal agranular cortex in vitro.

A slice preparation of rat frontal agranular cortex preserving commissural inputs has been used for intracellular recording from layer V pyramidal cells, in order to characterize the synaptic potentials induced by stimulation of the corpus callosum and to reveal the subtypes of amino acid receptors involved. Stimulation of the corpus callosum induced EPSPs followed by early IPSPs with a peak latency of 30 +/- 2 ms and late IPSPs with a peak latency of 185 +/- 18 ms. Reversal potentials for early and late IPSPs were -75 +/- 5 mV (early) and -96 +/- 5 mV (late). Late IPSPs were more dependent on extracellular K+ concentration. The early IPSPs were blocked by GABAA antagonists, bicuculline and picrotoxin, whereas the late IPSPs were reduced by the GABAB antagonist, phaclofen. CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), an antagonist of non-NMDA (N-methyl-D-aspartate) receptors, suppressed both EPSPs and late IPSPs at 5 microM. Early IPSPs remained at this concentration but were suppressed by 20 microM CNQX. In Mg(2+)-free solution, EPSPs were larger and more prolonged than in control solution. These enhanced EPSPs persisted after 5 to 20 microM CNQX, but were reduced in amplitude, and their onset was delayed by 3.6 +/- 0.8 ms. The remaining EPSPs were suppressed by 50 microM APV (DL-2-amino-5-phosphono-valeric acid), an antagonist of NMDA receptors. In Mg(2+)-free solution containing 5 to 20 microM CNQX, the late IPSPs were not diminished. The remaining late IPSPs were suppressed by APV or by phaclofen.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1987

N-methyl-D,L-aspartate elicits hypothalamic gonadotropin-releasing hormone release in prepubertal male rhesus monkeys (Macaca mulatta).

In higher primates, the protracted delay from infancy to puberty results from an interruption in hypothalamic GnRH release. To determine whether the quiescent hypothalamic GnRH neurons of the prepubertal macaque are capable of discharging the decapeptide in response to a generalized neural depolarization, an excitatory amino acid analog, N-methyl-D,L-aspartate (NMA), was administered systemically to orchidectomized rhesus monkeys between 13 and 20 months of age. GnRH secretion was estimated indirectly by monitoring changes in circulating LH concentrations after the responsivity of pituitary gonadotropes to GnRH had been greatly facilitated by the chronic intermittent iv infusion of GnRH (0.1 microgram/min for 3 min every hour). The iv bolus administration of increasing doses of NMA (1.5, 4.8, and 15.0 mg/kg BW), 10-14 h after termination of the priming infusion of GnRH, elicited distinct discharges of LH, with magnitudes directly related to the amount of the excitant injected. Administration of a higher dose of NMA (48 mg/kg BW), however, failed to induce further LH release. The finding that pretreatment with a long-acting and potent GnRH receptor antagonist [( AcD2Nal1,4ClPhe2,DTrp3,DArg6,DAla10] GnRH-HOAc) abolished the LH-releasing activity of NMA provides compelling evidence for the view that the action of the neural excitant to induce gonadotropin release was exerted at a suprapituitary level. The additional observation that an N-methyl-D-aspartate receptor antagonist (D,L-2-amino-5-phosphono-valeric acid) blocked the NMA-induced release of GnRH suggests that the amino acid analog interacted with the N-methyl-D-aspartate receptor on neurons that synthesize and/or control the release of the hypothalamic hormone. Most interestingly, three sequential GnRH discharges, with a period and an amplitude apparently similar to those generated by the hypothalamus of the adult, were elicited from the brain of prepubertal monkeys by the sequential administration of three injections of NMA at hourly intervals. Taken together these findings demonstrate that the apparent dormancy of hypothalamic GnRH neurons, which is characteristic of prepubertal development in higher primates and underlies the protracted delay in the onset of puberty in these species, may be readily terminated by application of a generalized neural excitation. Plasma FSH, PRL, GH, and cortisol concentrations were also monitored during the course of some of these experiments, and release of each of these four hormones was observed after the iv injection of NMA (15 mg/kg BW).

Animals↗
PubMed1994

Regulation of dopamine levels in intrastriatal grafts of fetal mesencephalic cell suspension: an in vivo voltammetric approach.

An in vivo voltammetric technique was used to monitor dopamine (DA) release in the 6-hydroxydopamine (6-OHDA)-lesioned rat striatum reinnervated by grafts of ventral mesencephalon containing DA neurons. Extracellular levels of DA were measured during the administration of D1 or D2 DA receptor antagonists. In addition, changes in DA levels induced by agonists and antagonists of excitatory amino acid (EAA) receptors were studied to verify the possible existence of a host glutamatergic control on the grafted DA cells in the 'transplanted' rats. Two months after the grafts were performed, the voltammetric signal measured under baseline conditions in the grafted striata was found to be almost similar to that recorded on the contralateral control side. Likewise, in another group of transplanted rats, the turnover of the amine, as expressed by the DO-PAC/DA tissue level ratio, was found to have become "normalized" after grafting, compared with the lesion-only group. The increase in the voltammetric signal observed after administering the D2 antagonist sulpiride (100 mg/kg i.p.) was lower in the grafted striata than on the contralateral side, however. This suggests that some D2 autoreceptor subsensitivity may have helped to maintain the baseline level of dopaminergic transmission. Adaptive processes of this kind might compensate for the partial DA reinnervation of the host striatum found to occur on the basis of the tyrosine hydroxylase immunostaining patterns. After administration of either the D1 antagonist SCH 23390 (0.1 mg/kg s.c.), or injection of EAA receptor agonists--1-glutamate, quisqualate and N-methyl-D-aspartate (all 10 nmol i.c.v.)--and antagonists--amino-phosphono-valeric acid (10 nmol i.c.v.) and dizocilpine (MK801, 0.2 mg/kg i.p.)--no significant differences between the two striata were detected in the voltammetric signals. These results suggest that, in the grafted rats, neurons belonging to the host population, such as the striatal cells bearing D1 receptors or the corticostriatal afferents presumed to contain glutamate, might modulate the DA levels, as was found to occur under normal conditions.

2-Amino-5-phosphonovalerate↗
PubMed2006

Performance responses and indicators of gastrointestinal health in early-weaned pigs fed low-protein amino acid-supplemented diets.

The effects of low-protein AA-supplemented diets on piglet performance, visceral organ mass, incidence of diarrhea, intestinal microbial population, and fermentation were studied in a 3-wk trial. After a 7-d adaptation period, 96 piglets (approximately 6.2 kg of initial BW) were assigned to 4 corn-wheat, soybean meal-based dietary treatments in a completely randomized design to give 6 replicate pens per treatment (n = 4 piglets per pen). The treatments were a control wheat-corn-soybean meal-based phase I diet containing 23% CP, or the same diet with CP reduced to 21%, 19%, or 17% and supplemented with crystalline AA to achieve equal standardized ileal digestible contents of Lys, Met plus Cys, Thr, and Trp in all diets. Diets were formulated to similar nutrient levels and provided ad libitum. Blood from all pigs was taken on d 0, 7, 14, and 21 for determining plasma urea N. Weekly feed intake, BW changes, and G:F were determined. On d 21, 2 pigs per pen were randomly selected and killed to determine small intestinal morphology, digesta pH and ammonia levels, and luminal microbial counts. Average daily feed intake, ADG, and G:F were not affected (P > 0.10) by reducing CP to 21%, but a reduction to 19% or 17% decreased ADFI (P < 0.001) and ADG (linear, P < 0.001; quadratic, P < 0.05) over the 3-wk study period. Reducing CP to 19% had no effect (P > 0.10) on G:F; however, this response criterion was decreased linearly (P < 0.001) over the 3-wk study period as dietary CP declined. Water usage was only numerically decreased (P > 0.10) with dietary CP reduction. Plasma urea N was decreased linearly (P < 0.01) with CP reduction. Reducing CP from 23 to 17% had a linear (P < 0.05) and cubic effect on stomach and liver weights, respectively. Although histological data showed some differences among diets, no distinct trend was evident. Ammonia N in ileal digesta was reduced linearly (P < 0.01) as dietary CP was decreased. With the exception of valeric acid, VFA levels in ileal digesta of piglets fed low-protein diets were generally lower (P < 0.05) compared with the control diet. Diet had no effect on intestinal microbial counts (P > 0.10). The results show that piglet performance may suffer when dietary CP is reduced by 4 or more percentage units from 23% and support the hypothesis that low-CP diets help maintain enteric health in pigs by lowering toxic microbial metabolites such as ammonia.

Amino Acids↗
PubMed1998

Calcium phosphate induction by sol-gel-derived titania coatings on titanium substrates in vitro.

Titanium and its alloys are used widely in the manufacture of orthopedic and dental implants. Sol-gel-prepared titania is able to stimulate bone-like apatite formation in in vitro and in vivo cultures. These materials can be used, for example, as coatings on dental and orthopedic implants. However, the processes that lead to apatite formation are not fully understood. In this study different kinds of titania coatings on commercially pure titanium (c.p. Ti) were tested for apatite-forming ability. The rate of apatite formation is considered to be descriptive of a material's bioactive (bone-bonding) potential. Apatite-forming tests were done in simulated body fluid (SBF). Apatite-forming ability was highest with the addition of valeric acid to sol (600 degrees C) or with sintering sol-gel coatings at 450 degrees-550 degrees C. At that temperature range calcium phosphate forms on the coatings in 1 week. Calcium phosphate forming is observed in 1 day on standard coatings sintered at 500 degrees C.

Biocompatible Materials↗
PubMed2003

Inhibition of alpha-ketoglutarate dehydrogenase complex promotes cytochrome c release from mitochondria, caspase-3 activation, and necrotic cell death.

Mitochondrial dysfunction has been implicated in cell death in many neurodegenerative diseases. Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key and arguably rate-limiting enzyme of the Krebs cycle, occurs in these disorders and may underlie decreased brain metabolism. The present studies used alpha-keto-beta-methyl-n-valeric acid (KMV), a structural analogue of alpha-ketoglutarate, to inhibit KGDHC activity to test effects of reduced KGDHC on mitochondrial function and cell death cascades in PC12 cells. KMV decreased in situ KGDHC activity by 52 +/- 7% (1 hr) or 65 +/- 4% (2 hr). Under the same conditions, KMV did not alter the mitochondrial membrane potential (MMP), as assessed with a method that detects changes as small as 5%. KMV also did not alter production of reactive oxygen species (ROS). However, KMV increased lactate dehydrogenase (LDH) release from cells by 100 +/- 4.7%, promoted translocation of mitochondrial cytochrome c to the cytosol, and activated caspase-3. Inhibition of the mitochondrial permeability transition pore (MPTP) by cyclosporin A (CsA) partially blocked this KMV-induced change in cytochrome c (-40%) and LDH (-15%) release, and prevented necrotic cell death. Thus, impairment of this key mitochondrial enzyme in PC12 cells may lead to cytochrome c release and caspase-3 activation by partial opening of the MPTP before the loss of mitochondrial membrane potentials.

Animals↗
PubMed1989

High-pressure liquid chromatographic analysis of eflornithine in serum.

Eflornithine [2-(difluoromethyl)-DL-ornithine monohydrochloride, monohydrate; DFMO] is an ornithine decarboxylase inhibitor used to treat Pneumocystis carinii pneumonia (PCP) in AIDS patients unresponsive or intolerant to conventional drug therapy. A reversed-phase HPLC method utilizing precolumn dansylation is described which permits the analysis of DFMO in serum of AIDS patients in support of pharmacokinetic studies. Norvaline (2-amino valeric acid) was added as the internal standard and the sample was extracted with three portions of ice-cold 80% ethanol. The supernatants were evaporated in a vacuum oven at 50 degrees C, and the residue was dissolved in base and derivatized at 25 degrees C for 4 h in the dark using dansyl chloride in acetone. Dilute base was then added and 25 to 50 microL was injected into the HPLC. Chromatography was performed on a C-8, 15-cm column with a linear gradient from a 95:5 solution of 10 mM pH 4 acetate buffer and THF, to a 90:10 solution of acetonitrile and THF over a period of 28 min. Detection was by UV at 330 nm, and DFMO and norvaline eluted at 14.7 and 17.7 min, respectively. The method was linear over a range of 5 to 950 micrograms/mL, and replicate analyses of a 25-micrograms/mL control specimen produced a between-run coefficient of variation of 6%. No interferences were encountered in a variety of patient and normal serum blanks.

Acquired Immunodeficiency Syndrome↗