Folate-homocysteine interrelations: potential new markers of folate status.

We report a transient drop in plasma Hcy and Cys following a single oral dose of PteGlu. The thiol change was concomitant with both the peak plasma 5CH3H4PteGlu1 level (by HPLC) and the maximum plasma Lactobacillus casei activity which reflects absorption of unmodified PteGlu. The significant reciprocal association of Hcy with radioassay RBC folate (r = -0.28, 99% CI -0.48, -0.05, P = 0.0016), serum folate (r = -0.37, 99% CI -0.56, -16, P = 0.0001), and vitamin B12 (r = -0.42, 99% CI -0.59, -21, P = 0.0001) is shown and reflects the long-term nutritional effect of B vitamins on this important, potentially atherogenic thiol. These are now well-established associations. We extend the potential for investigation of folate metabolism in health and disease by evaluating a range of new folate indices which are based on erythrocyte coenzymes. These have been looked at independently and in association with established parameters. Erythrocyte methylfolates (mono- to hexaglutamate-5CH3H4PteGlu1-6), formylfolates (tri- to pentaglutamate-5CHOH4PteGlu3-5),formiminotetrahydrofolate (formiminoH4PteGlu1), unsubstituted tetrahydrofolate (H4PteGlu1), andpara-aminobenzoylglutamate (P-ABG) have been measured by HPLC with fluorescence detection. A positive linear association exists between (i) H4PteGlu1 and radioassay RBC folate (r = 0.50, 99% CI 0. 07, 0.77, P = 0.0036), and (ii) H4PteGlu1 and tetraglutamates of both formyl- and methylfolate (r = 0.52, 99% CI 0.10, 0.78, P = 0. 0022, and r = 0.56, 99% CI 0.15, 0.80, P = 0.0009, respectively). Since, in addition, a reciprocal linear association exists between Hcy and tetraglutamyl formylfolate (r = -0.41, 99% CI -0.73, 0.05, P = 0.0206), erythrocyte tetraglutamates may be a good reflection of the bodies' active coenzyme pools. Pentaglutamyl formylfolate, the longest oligo-gamma-glutamyl chain form of this coenzyme may be a good indicator of folate depletion. The abundance of this coenzyme both increases with increasing Hcy (r = 0.55, 99% CI 0.13, 0.80, P = 0.0014) and increases as H4PteGlu1, the principle folate congener, decreases (r = -0.59, 99% CI -0.82, -0.20, P = 0.0004). Furthermore, the apparent equilibrium between substrate (5CH3H4PteGlu1) and product (H4PteGlu1) of methionine synthase is significantly associated with the abundance of 5CHOH4PteGlu5 (r = -0.53, 99% CI -0. 79, -0.11, P = 0.0018). This suggests that low methionine synthase activity for whatever reason (metabolic or dietary) may lead to an increase in the relative abundance of 5CHOH4PteGlu5. Like 5CHOH4PteGlu5, evidence is given that 5CH3H4PteGlu6, the longest oligo-gamma-glutamyl chain form of this particular coenzyme pool, may also be a good indicator of folate depletion. This is shown by a change in the relative proportion of erythrocyte methylfolate polyglutamates following supplementation with 400 microg/day PteGlu. Short-chain polyglutamates of methylfolate (5CH3H4PteGlu1--> 5CH3H4PteGlu4) increase in proportion to the total methylfolate pool, while long-chain polyglutamates of methylfolate (5CH3H4PteGlu5 and particularly 5CH3H4PteGlu6) decrease in their relative abundance.

Impaired regeneration of monoglutamyl tetrahydrofolate leads to cellular folate depletion in mothers affected by a spina bifida pregnancy.

Periconceptional folate prevents neural tube defects (NTD) by a mechanism which is unclear. The present study found significant changes in the equilibrium of the homocysteine remethylation cycle in NTD affected mothers, possibly involving B12-dependent methionine synthase or 5,10-methylenetetrahydrofolate reductase. Data were consistent with impaired Hcy remethylation leading to poor regeneration of H4PteGlu1, the main intracellular precursor of all folates. This lesion leads to cellular folate deficiency indicated by a significantly lower radioassay RBC folate and 5CH3H4PteGlu4 in affected mothers. The drop in this tetraglutamate is associated with an increase in the abundance of longer chain oligo-gamma-glutamyl folate, again reflecting the underlying folate deficiency. This effect may compromise purine, DNA-thymine, and methionine production, particularly during embryogenesis when folate demand is high. At this time serine hydroxymethyltransferase may play a critical role in conserving H4PteGlu1 for purine synthesis. Many of these depletion effects were corrected with folate supplementation for 1 month.

Risk of neural tube defect-affected pregnancy is associated with a block in maternal one-carbon metabolism at the level of N-5-methyltetrahydrofolate:homocysteine methyltransferase.

The disposition of whole blood mono-to hexaglutamyl methylfolate and plasma homocysteine (HCY) was used to evaluate potential lesion sites in one-carbon metabolism which could be responsible for neural tube defect(NTD)-affected pregnancies. An isocratic high-performance liquid chromatographic system (HPLC) with photodiode array detection was used to quantify and speciate whole-blood methylfolate into mono-, di-, tri-, tetra-, penta-, and hexaglutamate forms. This technique was also used with off-line radioassay to identify nonmethyl whole-blood folates. Isocratic HPLC with fluorescence detection was used to quantify SBDF derivatized homocysteine in plasma. The study investigated blood from 11 women who had experienced a previous NTD-affected pregnancy and 11 controls of similar age and social class. No subjects were pregnant. HCY levels were significantly higher in NTD subjects (P = 0.0486, 95% CI-2.799,0.001 using the Mann-Whitney test), as was the ratio of known intracellular (tri-to hexaglutamyl) methylfolate compared to extracellular (mono- and diglutamyl) methylfolate (P = 0.0062 95% CI-0.543, 3.862 using the Mann-Whitney test). Vitamin B12, red cell folate, circulating total methylfolate, and circulating mono-to hexaglutamyl methylfolates showed no difference between population groups. The disposition between individual and cumulative glutamate chain lengths of methylfolate showed significant trends which differed between population groups: (i) total blood methylfolate (Glu1-6) appears to be utilized by N-5-methyltetrahydrofolate:homocysteine methyltransferase (MS) in control blood but not NTD blood, where it appears to accumulate following a 45-min incubation; (ii) whole-blood hexaglutamyl methylfolate (5CH3-H4PteGlus) becomes a larger proportion of the total blood methylfolate in NTD than in control populations; and (iii) the intermediate glutamate chains of methylfolate (Glu1-5) remain relatively constant as 5CH3-H4PteGlu6 accumulates in NTD but appear to increase linearly with 5CH3-H4PteGlu6 in controls. The significant elevation of HCY in the NTD population is associated with the increasing proportion of 5CH3-H4PteGlu6 relative to the total methylfolate, since, when corrected for HCY level, the proportion of 5CH3-H4PteGlus to total methylfolate is similar in NTD and control populations. These trends are consistent with a defect at the level of vitamin B12 dependent MS which "traps" folate at the 5CH3-H4PteGlus level.

The influence of dietary folate and methionine on the metabolic disposition of endotoxic homocysteine.

We have investigated the disposition of potentially endotoxic homocysteine (Hcy) and its transsulfuration metabolite cysteine (Cys) in 98 individuals (age range 20-66 years). Our study reports on the relationship between Hcy and two important dietary factors likely to influence plasma levels of this thiol: dietary folate and dietary methionine. chi2 analysis shows a low frequency of elevated plasma Hcy at high folate intake. This frequency for Hcy >10 micromol/liter with a folate intake >350 microg/day is significant (P < 0.02). The data reflect a tendency for elevated Hcy values to be associated with low dietary folate, although many subjects with a low dietary folate also had a low plasma Hcy. Intake of dietary methionine was found to be significantly higher in males than in females (P < .0001). This may account for the looser relationship between Hcy and its transsulfuration product, Cys, in females (R2 = 0.30) compared to males (R2 = 0.73), since conversion of methionine to SAM in males would activate cystathionine beta synthase and commit excess Hcy to transsulfuration. The generally lower methionine intake of females means that more Hcy is utilized in the remethylation cycle in which methionine is produced from the de novo methyl group of 5-methyltetrahydrofolate or from the preformed methyl group of betaine. Clearly a Hcy moiety locked up in remethylation would be further removed from Cys, the end product of transsulfuration. An increasing number of studies are clarifying the relationship between Hcy, folate, and other B vitamins. However, less attention seems to be given to the influence of dietary methionine on the disposition of Hcy. The present study supports biochemical theory and indicates that more focus should be given to the effect of dietary methionine on Hcy. These findings have particular significance since even moderate increases in plasma Hcy are associated with a toxic vascular effect. Consequently the relationship between dietary folate and Hcy levels should be a factor in evaluating recommended dietary allowances for this vitamin. The simplicity of our dietary folate questionnaire also raises the possibility of a screening test in which individuals can ascertain whether their folate intake is adequate to reduce Hcy levels to a benign value.

Determination of plasma total homocysteine and cysteine using HPLC with fluorescence detection and an ammonium 7-fluoro-2, 1, 3-benzoxadiazole-4-sulphonate (SBD-F) derivatization protocol optimized for antioxidant concentration, derivatization reagent concentration, temperature and matrix pH.

A sensitive HPLC-fluorescence method for determining total endogenous plasma homocysteine (Hcy), cysteine (Cys) and cysteinylglycine (Cys-Gly) following derivatization with ammonium 7-fluoro 2,1,3-benzoxadiazole-4-sulphonate (SBD-F) is described. Quantitation utilizes an internal standard, 2-mercaptoethylamine. The derivatization procedure has been optimized for concentration of SBD-F, reducing agent (tributylphosphine) and temperature. Findings indicate that values for plasma determinations vary according to the nature of the matrix in which calibration standards are made up. If quantitation is based on a peak height ratio, then standards should be made up in either pH 7.4 phosphate buffered saline or plasma taking into account the endogenous thiol concentration. These findings are based on calibration data, and 30 plasma samples quantified using thiol standards made up in plasma, pH 7.4 and pH 9.5 buffers. By defining how this matrix/pH effect influences thiol quantitation, it should be possible to make a more meaningful comparison of Hcy measurements between laboratories. The chromatographic separation was investigated at several mobile-phase pH values with the following conditions ascertained to be optimal: a mobile phase consisting of 5% (v/v) acetonitrile in 0.1 M KH2PO4, pH 2.15 was run at a flow rate of 0.5 mL/min. It was used in conjunction with a Supelco LC-18 base deactivated analytical column (150 x 4.6 cm i.d. 3 microM bonded silica). The internal standard and thiols were measured by fluorescence detection at 385 nm excitation and 515 nm emmission. Plasma levels are easily measured in a 100 microL volume. Storage for 2 months at -20 degrees C resulted in no deterioration of thiols. Furthermore, no difference in thiol levels was observed between bloods collected in lithium heparin and EDTA. Collected blood should, however, be separated as soon as possible to avoid red cell metabolism of Hcy which was observed in a case of hyperhomocysteinemia. Once derivatized, thiols are stable for at least one week at +4 degrees C.

Analysis and biochemistry of blood folate.

Although the analysis of low plasma concentrations of 5-methyltetrahydrofolate by several specific HPLC methods has been reported, considerably fewer routine chromatographic techniques exist for the analysis of specific folate coenzymes in the erythrocyte where a nonspecific bioassay indicates that the vitamin achieves a level 10 times higher than that in plasma. By using three separate folypolyglutamate deconjugation procedures and combining an extraction technique which adequately preserves all native folate coenzymes with an HPLC technique utilizing fluorescence, diode array, and off-line radioassay detection capable of resolving all crucial native folates in their monoglutamyl forms, we were unable to demonstrate levels of 5-methyltetrahydrofolate in whole blood hemolysate beyond what might be expected from the plasma component. While the exact nature of erythrocyte folate could not be ascertained, we provide evidence that a proportion of it may exist at the formyl level of oxidation. The complex pH and enzymatic interrelationship between folate coenzymes at the formyl oxidation level is discussed in terms of our extraction technique and findings, as well as in a broader biological context. This paper also describes a simple acid precipitation technique for measuring plasma 5-methyltetrahydrofolate, as well as providing comprehensive data on the chromatographic behavior of all the folylmonoglutamates in reversed-phase and weak anion-exchange modes, including useful spectral data for optimizing detection parameters and identifying individual coenzymes. 10-Formyltetrahydrofolate and 5-methyltetrahydrofolate are the two most important one-carbon-substituted folate coenzymes. 10-Formyltetrahydrofolate is unavailable commercially, probably due to its instability. We chart the chemical synthesis of this important coenzyme and show that it and what is thought to be 5,10-hydroxymethylenetetrahydrofolate are actually minor products compared to the parent 5,10-methenyltetrahydrofolate and the ultimate reaction product, 5-formyltetrahydrofolate. Since intraerythrocyte folate binds to a specific hemoglobin site, we ascertained the total number of binding sites on hemoglobin (Bmax) and the equilibrium dissociation constant (Kd) for 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and the antimetabolite methotrexate. Binding affinities were consistent with a low-affinity, low-capacity interaction for all three. It was demonstrated that hemoglobin has a greater affinity for 5-methyltetrahydrofolate than for the other folate derivatives (Kd = 1.2 x 10(-3) M), while rather surprisingly, methotrexate had a higher affinity for hemoglobin than did 5-formyltetrahydrofolate (Kd = 2.5 x 10(-3) and 3.7 x 10(-2) M, respectively.

Nonenzymatic degradation and salvage of dietary folate: physicochemical factors likely to influence bioavailability.

We investigated the oxidative degradation pathway of 5CH3-H4PteGlu, the main extracellular folate and the predominant form of the vitamin found in food and blood. 5CH3-H4PteGlu is oxidized to 5CH3-5,6-H2PteGlu which subsequently undergoes C9-N10 bond cleavage yielding a pteridine residue and P-ABG, the latter step resulting in irreversible loss of vitamin activity. Under moderately acid conditions typical of the postprandial gut (pH 3.5) 5CH3-H4PteGlu is fairly stable (t1/2 = 273.6 min), while 5CH3-5,6-H2PteGlu is rapidly degraded (t1/2 = 16.9 min). In a neutral environment (pH 6.4) stability is reversed; 5CH3-H4PteGlu t1/2 = 12.0 mins, 5CH3-5,6-H2PteGlu t1/2 = 1504.6 min. Ascorbic acid was efficacious in the facile salvage of 5CH3-H4PteGlu from 5CH3-5,6-H2PteGlu which occurred rapidly and with significant efficiency (100% conversion) under acid (pH 3.5) conditions, t1/2 = 1.3 min (1 mmol/liter ascorbate), but was less efficient under neutral (pH 6.4) conditions t1/2 = 273.6 min (36% conversion). The presence of zinc and iron broadly maintains the pattern of effect, but increases all reaction rates. PteGlu was stable under all conditions studied. These results obtained in an artificial environment were supported by findings in human gastric juice: at a gastric pH of 1.47 with low endogenous ascorbate (7.0 mumol/liter), 5CH3-5,6-H2PteGlu and 5CH3-H4PteGlu both degrade instantly via C9-N10 bond cleavage to yield an equimolar amount of P-ABG. If the same gastric juice is spiked at 58.0 mumol/liter ascorbate (moderate endogenous concentration), 5CH3-H4PteGlu is stable (t1/2 = 334.7 min), while 5CH3-5,6-H2PteGlu is instantly salvaged to 5CH3-H4PteGlu with 43.3% efficiency, and the remaining 5CH3-5,6-H2PteGlu is degraded to P-ABG. In gastric juice with an elevated pH of 7.0 and no endogenous ascorbate, 5CH3-5,6-H2PteGlu and 5CH3-H4PteGlu are both stable, with no C9-N10 bond cleavage. This, for 5CH3-H4PteGlu, is in apparent contrast to findings at pH 6.4 in an artificial environment. The same gastric juice spiked to 50 mumol/liter ascorbate did not result in 5CH3-H4PteGlu salvage from 5CH3-5,6-H2PteGlu.(ABSTRACT TRUNCATED AT 400 WORDS)

Methylfolate modulates potassium evoked neuro-secretion: evidence for a role at the pteridine cofactor level of tyrosine 3-hydroxylase.

We have previously shown that 5-methyltetrahydrofolate influences neuro-secretion. The present study more precisely characterises the processes involved and considers one probable site of action. Focusing on the tyrosine-noradrenalin axis in cerebellum we showed 5-methyltetrahydrofolate causes a significant reduction in the apparent K+ evoked secretion of noradrenalin to only 12.9% of control release. Evidence supports the idea that this could actually be due to increased synthesis leading to; depletion of reserves, possibly through leakage, exocytotic inhibition via activation of presynaptic receptors or end product inhibition by noradrenalin at the pteridine cofactor level of tyrosine hydroxylase: a) concomitant decreased measurement of perfusate and intracellular tyrosine with released noradrenalin following 5-methyltetrahydrofolate treatment supports the idea of increased transmitter turn over; b) kinetic studies indicate that at saturating concentrations of tyrosine and in the presence of an inhibitor of L-DOPA decarboxylase, 5-methyltetrahydrofolate partially duplicates the rate limiting behaviour of a synthetic pteridine cofactor--DL,2-amino-4-hydroxy-6,7,dimethyltetrahydropteridine. We debate whether, in vivo, CSF 5-methyltetrahydrofolate might interact at the tetrahydrobiopterin cofactor level of tyrosine hydroxylase and other aromatic amino-acid hydroxylases.

The methylfolate axis in neural tube defects: in vitro characterisation and clinical investigation.

We have investigated various micronutrients important to folate metabolism in women with two previous neural tube defect (NTD)-affected pregnancies. Results suggest the disposition of plasma 5-methyltetrahydrofolate (5CH3-H4PteGlu) with respect to dietary intake may differ from that of the control population. It appears that to achieve a given plasma level of 5CH3-H4PteGlu, the population with a history of NTD pregnancies needs to take in more dietary folate than controls. We discuss this in the context of a potential lesion at or upstream from 5,10-methylenetetrahydrofolate reductase (MTHFR). This metabolic axis, which is responsible for the multienzymic conversion of PteGlu to 5CH3-H4PteGlu, has been investigated in a rat model using liver homogenate. The anticonvulsant drug (ACD) carbamazepine was found to inhibit the reaction in terms of a reduced Vmax and increased Km. Inhibition approaching maximal was found to occur at therapeutic levels of ACD. Various potential inhibitory sites along the methylfolate axis are considered and possible relationships to congenital malformations discussed. We describe folate and one carbon metabolism in relation to potential NTD lesion sites, not only in the light of present findings, but with respect to the published findings of other workers. Based on our hypothesis that an NTD lesion exists upstream from MTHFR, we expound how pteroylmonoglutamate supplementation may protect against NTD (i) by reducing endotoxic homocysteine and (ii) through inhibiting MTHFR (as do dihydrofolates) and thus diverting one carbon units into DNA thymine.

Modulation of potassium evoked secretory function in rat cerebellar slices measured by real time monitoring: evidence of a possible role for methylfolate in cerebral tissue.

The real time dynamics of K+ evoked neurosecretion in cerebellar slices has been monitored electrochemically. In the presence of 5-methyltetrahydrofolate a statistically significant diminution in secretory response occurs. Agonists to probe the pharmacological basis for this indicate it is not due to voltage sensitive Ca2+ channel blockade, nor does it show any similarity of effect with kainate, whose receptor is a putative binding site for 5-methyltetrahydrofolate. The method is fully validated, although no account is taken of individual molecular species. High performance liquid chromatography combined with off line microbiological assay could only detect 5-methyltetrahydrofolate in cerebrospinal fluid. We therefore discuss our findings in relation to possible cerebral roles for cerebrospinal fluid 5-methyltetrahydrofolate in the context of both membrane and transmitter related interactions.

Solid phase extraction of oxcarbazepine and its metabolites from plasma for analysis by high performance liquid chromatography.

A rapid, sensitive and simple-to-operate high performance liquid chromatographic method for the simulataneous determination of oxcarbazepine, 10-hydroxycarbazepine and 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octadecylsilane bonded-silica columns (Bond Elut C18, 1 mL capacity). Chromatographic separation of oxcarbazepine and its metabolites was achieved using a mobile phase consisting of acetonitrile/methanol/water (13:25:62 by volume) at a flow rate of 1.2 mL/min in conjunction with a Waters Associates Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a LiChrospher 5 microns C18 guard column. By measuring the UV absorbance at 214 nm, plasma levels in the region of 50-100 ng/mL for the drug and its metabolites can be detected with only 100 microL of plasma. The method has been applied to pharmacokinetic studies of oxcarbazepine and its metabolites in children with epilepsy; preliminary pharmacokinetic findings in two patients at steady-state are presented.

Daily variations in steady-state plasma concentrations of carbamazepine and its metabolites in epileptic children.

Total plasma carbamazepine, carbamazepine-10,11-epoxide (CBZ-EP) and 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine (CBZ-DIOL) concentrations were measured during a 24h period in 21 patients receiving carbamazepine monotherapy, in equally divided doses, every 12h. Interdose and diurnal variations in plasma concentrations of parent drug and metabolites were assessed. Carbamazepine and both metabolites showed significant differences in mean 4h post-dose plasma concentrations between day and night dosing (p less than 0.001). Significant linear correlations were obtained between carbamazepine dose and plasma concentrations of carbamazepine, CBZ-EP and CBZ-DIOL when sampling times were standardised (p less than 0.01). Comparisons of plasma concentrations of the parent compound with those of its 2 main metabolites revealed significant linear correlations in all cases (p less than 0.01). The effects of daily fluctuations in plasma concentrations of all 3 compounds on their relative concentrations (CBZ-EP:carbamazepine, CBZ-DIOL:carbamazepine and CBZ-DIOL:CBZ-EP) during the 24h period were also determined: the plasma concentration ratios CBZ-EP:carbamazepine and CBZ-DIOL:carbamazepine were significantly related to the dose of carbamazepine at fixed sampling times (p less than 0.05, with 1 exception). The large interdose and diurnal variation in plasma carbamazepine concentrations observed in this study (approximately 40% decrease from peak to trough) has important implications both clinically and in relation to therapeutic drug monitoring.

Factors influencing plasma level/dose ratios of carbamazepine and its major metabolites in epileptic children.

The relationship between daily dose and plasma concentrations of carbamazepine (CBZ), CBZ-10,11-epoxide (CBZ-EP), and 10,11-dihydro-10,11-trans-dihydroxy-CBZ (CBZ-DIOL) was investigated in 21 children aged 7-16 years who received CBZ monotherapy, twice daily in equally divided doses. Significant linear correlations between CBZ dose and plasma levels were obtained for CBZ and its metabolites (p less than 0.01). In addition, the effects of daily dose and patients' age on the plasma level/dose ratios for CBZ, CBZ-EP, and CBZ-DIOL were evaluated. A significant negative correlation was observed between the daily dose of CBZ and the CBZ plasma level/dose ratio (p less than 0.01). By contrast, plasma level/dose ratios for CBZ-EP and CBZ-DIOL were independent of dose (p greater than 0.1). On the basis of these observations, we consider that the decrease in CBZ plasma level/dose ratio with increasing CBZ dose appears to be due to dose-dependent metabolic clearance of CBZ. The influence of age on plasma level/dose ratios for CBZ and its metabolites was not significant (p greater than 0.05). However, there was considerable interdose and diurnal variation in the plasma level/dose ratios, particularly for CBZ (28-41%); this must be taken into account when making dose adjustments based on plasma level/dose ratios.

In vivo characterization of the absorption and biotransformation of pteroylmonoglutamic acid in man: a model for future studies.

HPLC-EC has been used to measure the appearance of 5-CH3-H4 folic acid in human plasma following oral administration of folic acid. The process was found to be saturable in accordance with Michaelis-Menten kinetics. The apparent Km for this enzyme system indicates that low doses of oral folic acid are rapidly converted into 5-CH3-H4 folic acid, an observation consistent with the needs of intestinal absorption of essential trace nutrients. The appearance of L. casei active folate in plasma was not rate-limited and showed a biphasic relationship to dose. Preparative HPLC combined with L. casei bioassay demonstrated that most of the L. casei active folate appearing in plasma following a 20,000-micrograms dose of folic acid was due to the unmodified vitamin, only 5.6% being due to 5-CH3-H4 folic acid and with no detectable contribution from 5-CHO-H4 folic acid. The absorption characteristics of the system seem consistent between and within subject(s). No relationship could be demonstrated between predose levels of plasma 5-CH3-H4 folic acid and total folate in erythrocytes, which reflect the status of transport and storage forms of the vitamin, respectively.

A rapid and specific HPLC-electrochemical method for the determination of endogenous 5-methyltetrahydrofolic acid in plasma using solid phase sample preparation with internal standardization.

A rapid and specific HPLC-electrochemical method for determining endogenous 5-methyltetrahydrofolic acid (5MeTHF) in plasma is described. Quantitative solid phase extraction of 5MeTHF and internal standard, beta-hydroxyethyltheophylline, was carried out using proprietary phenyl bonded-silica columns (Bond Elut Phenyl cartridges, 1.0 mL capacity). Chromatographic separation was achieved using a mobile phase consisting of 15% (v/v) methanol in 0.05 M KH2PO4, pH 3.5 at a flow rate of 2.0 mL/min in conjunction with a Waters Assoc. radially compressed Nova-Pak phenyl column (10 cm x 8 mm, 4 microns bonded silica). The internal standard was measured by UV detection at 254 nm. A Bioanalytical Systems Inc. LC-17 glassy carbon oxidative flow cell with a potential held at +0.35 V vs Ag/AgCl using the LC-4A amperometric controller allowed levels of 1-2 ng/mL 5MeTHF to be measured in 500 microL of plasma. Daily appraisal of the ratio produced by authentic materials clearly demonstrated that quantitation using dual detection was not subject to problems of differential response. Inter-day variation of the differential detector response is cited. Comparison of the Lactobacillus casei bioassay with HPLC demonstrates good agreement between methods but at the same time highlights the drawback of using such non-specific methods to measure samples where more than one folylmonoglutamate may be present. Antoxidant free storage for three months at -70 degrees C in darkness resulted in no deterioration of 5MeTHF. A comparison of the means and range of values for plasma folate obtained using HPLC, L. casei bioassay and the radiometric binding assay is reported.

A rapid HPLC method for monitoring plasma levels of caffeine and theophylline using solid phase extraction columns.

A simple HPLC method for the determination of caffeine and theophylline in plasma is described. Separation of theobromine, paraxanthine, theophylline, beta-hydroxyethyltheophylline and caffeine is obtained using a mobile phase of 1% acetic acid/methanol (83:17, v/v) and a Waters Associates NOVA-PAK C18 column protected by a Guard-PAK precolumn module containing a Guard-PAK CN cartridge. Rapid sample preparation is achieved by solid-phase extraction columns (Bond-Elut C18, 1 mL capacity) which provide excellent recovery values for both drugs. The cost per sample using this approach can be minimised by column regeneration and re-use. Results obtained for theophylline are in good agreement with values determined by other techniques.