Circadian and Ultradian Rhythmicities in Very Premature Neonates Maintained in Incubators

Six very premature babies (born at 26–28 weeks gestational age) have been studied in hospital for 11–17 weeks, while in intensive care and in an incubator. Apart from suffering occasionally from the neonatal disorders of haemolytic jaundice and ‘respiratory distress of the newborn’, the babies were healthy and developed normally. Initially, the babies were continuously fed intravenously, and the lighting in the ward was on continuously. Routine care was given round the clock. When their medical condition permitted it, the babies were moved in their incubator to an adjacent ward, where they took frequent (2–4 hourly) small meals by mouth, the lighting was dimmed at night, and routine care tended to be given more in the daytime. Hourly recordings of insulated skin temperature were taken throughout the study, and it is the detection of rhythmicity in these measurements that has been the subject of the present study. The methods used were Phase-weighted Stacks, Phasor Walkout and Power Spectral Analysis. These methods have previously been used mainly in geophysical studies, and their value is that they can detect weak signals in noisy data and do not assume a particular waveform of any signal. Circadian rhythmicity was found in all babies for much of the time that were in the constant environment provided by the incubator. Ultradian rhythms were sometimes present also, but they were shorter-lived, and showed a wide range of changing periods, generally in excess of 8 h. When the babies were being treated for jaundice or respiratory distress, there was a tendency for the circadian rhythms to become weaker and for a broader spectrum of ultradian periods to appear. Placing babies in the 12 h : 12 h light : dark environment provided by the ward, and instituting feeding by mouth, had, in most cases, only modest effects upon either circadian or ultradian rhythms. Thus, circadian rhythms continued (but generally with a period not exactly equal to 24 h), and ultradian rhythms, when present, often did not show periods that could be related easily to feeding or care-giving. These results are discussed in terms of evidence for endogenous and exogenous origins of the observed rhythms, and of theories that have postulated the relationship between circadian and ultradian rhythms. It is concluded that the results from the present analyses are difficult to reconcile with the view that circadian rhythms develop from interactions between ultradian oscillators. We suggest that they indicate a matu-ration of the circadian system as a consequence of increasing associations between the circadian elements that are present in the suprachiasmatic nuclei and in other oscillators of the circadian system. The new analytical methods used here also indicate that the results obtained from time-frequency analysis depend to some extent upon the method used.     

Plasma concentrations of temazepam,a 3-hydroxy benzodiazepine,determined by electron-capture gas—liquid chromatography

A simple and sensitive high-performance liquid chromatographic assay of methotrexate (MTX) and its two active metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-di-amino-N¹⁰-methylpteroic acid (APA) in plasma, saliva and urine was developed. The method involved deproteinization with acetonitrile followed by addition of isoamyl alcohol and ethyl acetate. After extraction the sample was chromatographed on a cation-exchange column and monitored at 313 nm. The retention times were 5, 7 and 9 min and detection limits 20, 10 and 5 ng/ml for 7-OH-MTX, MTX and APA, respectively. For concentrations greater than 100 ng/ml one-step deproteinization of 0.1 ml sample with 0.25 ml acetonitrile was satisfactory for sample preparation. The method has been evaluated in samples from patients and rabbits receiving MTX.     

Cytochrome P450cam-catalyzed oxidation of a hypersensitive radical probe.

trans-1-Phenyl-2-vinylcyclopropane, a hypersensitive radical probe, is oxidized by cytochrome P450cam (CYP101) to a diastereomeric mixture of the corresponding epoxide (81%), (trans-2-phenylcyclopropyl)acetaldehyde (6%), and trans-5-phenyl-2-penten-1,5-diol (13%). trans-5-Phenyl-2-penten-1-ol and (trans-2-phenylcyclopropyl)ethane-1,2-diol are not detectably formed. Authentic standards of all the products have been synthesized and used to establish the identities (or the absence) of the metabolites. Studies with [18O]H2O demonstrate that the oxygens at positions 1 and 5 in the rearranged diol derive from molecular oxygen and water, respectively. Catalytic turnover of the enzyme is required for product formation from the olefin, but incubation of the epoxide metabolite with the enzyme, or with buffer alone, yields both the aldehyde and the rearranged diol products. The absence of trans-5-phenyl-2-penten-1-ol implies that the lifetime of the putative radical intermediate is so short that its existence as a discrete entity is questionable. A cationic intermediate is unlikely but cannot be excluded because the same metabolites are formed in a secondary reaction, even at pH 8.0, from the epoxide. The results provide no evidence for the involvement of radicals or cations in the epoxidation reaction, in agreement with results on the oxidation of olefins in organic solvents by metalloporphyrin catalysts.     

Rapid group separations of nucleotides and related compounds on silica columns

Nucleosides, bases, and nucleotides can be separated from one another rapidly (10–15 min) on 1-ml silica cartridges. Samples adjusted to 4 mm ammonium borate, 90% acetonitrile are loaded onto 1-ml columns equilibrated with the same solvent. Bases do not absorb to the silica under these conditions. Nucleosides are eluted with 16 ml of 0.5 m acetic acid in 90% acetonitrile. Nucleotides are then eluted with water. The 1-ml silica columns have performed well with samples up to 10 ml in volume. We have found the procedure to be quantitative and the gels to have high capacity (61 μmol Cyd/ml silica). Acid extracts from a large number of cells (10⁸) have been processed on a single cartridge.