M.I. Levene

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.

Optimisation of chromatographic conditions for the determination of folates in foods and biological tissues for nutritional and clinical work

Abstract Recent studies implicate folate metabolism in the aetiology of heart disease, neural tube defects, malignant transformation and affective disorders. The paper reports a rapid, isocratic HPLC separation of 11 folylmonoglutamate compounds which should prove useful when adapted to the varied needs of analysts researching these and other specific areas. Also reported are the separation, UV spectra including λmax values, fluorescence emission scans at an excitation wavelength of 295 nm and electrochemically derived hydrodynamic voltammograms with optimum oxidation voltages for p-aminobenzoylglutamate, tetrahydrofolate, 5-methyldihydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, dihydrofolate, pteroylmonoglutamate and 5,10-methylenetetrahydrofolate. In particular, 5-methyltetrahydrofolate, the main food folate and form of the vitamin found in plasma, can be measured easily by electrochemical detection using a low and highly selective voltage of 450 mV. This reduced folate is also readily detected fluorimetrically using an excitation wavelength of 295 nm and measuring emission at 365 nm. Electrochemical and fluorimetric detection offer equal sensitivity for 5-methyltetrahydrofolate measurement (300 pg on column). No other folate studied could be measured down to this level using fluorimetric detection under the described conditions. At pH 3.5, folate coenzyme λmax for UV detection varies between 267 and 300 nm with 5,10-methenyltetrahydrofolate giving maximum absorption at 355nm. UV measurement of 5-methyltetrahydrofolate is approaching an order of magnitude less sensitive than the former methods of detection. However, for in vitro studies, particularly in the form of a photodiode array, UV detection is a particularly useful tool. For cerebrospinal fluid, plasma, erythrocyte or food measurement of 5CH3H4PteGlu, electrochemical or fluorimetric detection is recommended; whilst for pharmacokinetic studies of plasma 5CHOH4PteGlu during methotrexate rescue therapy, electrochemical or UV detection is most appropriate. For analysis of plasma PteGlu following supplementation, or in food stuffs, UV detection offers the best measurement technique. The information presented should help address the major problem of trace folate analysis by HPLC, that is the need to combine high sensitivity with optimum selectivity in studying complex matrices such as physiological fluids, tissue preparations and food samples.

Minimizing the discomfort of neonatal intensive care

Abstract Pain is an ubiquitous experience in immature infants receiving intensive care and is an important, but not sole, source of stress among these babies. Pain and stress appear to have an effect on behaviour and pain response later in life. It has been shown that appropriate analgesia with opiates reduces the pain response and that this might have a beneficial effect on the childs response to pain later in life. Although there is insufficient evidence at the present time to be certain of the beneficial effects of analgesia/sedation, opiates appear to be safe, and current best evidence supports the use of continuous infusions of diamorphine or morphine during neonatal intensive care.

Student feedback: influencing the quality of teaching in a paediatric module

The aim of this study was to assess the impact of feedback on the quality of tutorials. Students completed structured feedback questionnaires on the perceived usefulness of teaching sessions. They perceived significant differences in the quality of tutorials delivered by experienced and inexperienced teachers (P < 0.01), although the differences tended to become less throughout the year. In the second year, teachers had advice from an experienced tutor in planning the sessions, in addition to receiving feedback from the students. During the second year, the students did not perceive any difference in the usefulness of these tutorials to them. The study showed that the quality of tutorials can be improved by passing structured feedback to the teachers, but that some skills training was also required to completely eliminate perceived differences.

Methylfolate exhibits a negative in-vitro interaction with important dietary metal cations

Abstract Methylfolate is an important dietary micronutrient essential for cell maturation and replication. Using HPLC with electrochemical detection we have shown that, at approaching neutral pH, anionic methylfolate complexes with metal cations leading to either oxidative degradation or possibly precipitation, the order of the effect being Zn2+>Ca2+ ≈ K+>Mg2+ ≈ Na+. Equimolar Zn2+ and Na+ enhance methylfolate decay 33.3- and 7.2-fold, respectively, when compared to the decay in water alone. This effect does not occur in the presence of reduced glutathione or at midly acid pH. These results may have important implications in vivo, particularly with respect to the bioavailability of dietary and pharmacological forms of the vitamin.