Variation in maternal urinary cortisol profiles across the peri-conceptional period: a longitudinal description and evaluation of potential functions.

STUDY QUESTION: How do women's first morning urinary cortisol levels, a marker of stress axis activity, vary during the peri-conceptional period (the 12 weeks around conception)? SUMMARY ANSWER: First morning urinary cortisol follows an overall increasing trajectory across the peri-conceptional period, interrupted by 2 week-long decreases during the week preceding conception and the fifth week following conception. WHAT IS KNOWN ALREADY: Later gestational stages (i.e. second and third trimesters) are characterized by increasing levels of circulating cortisol. This increase is hypothesized to constitute a response to the energy demands imposed by fetal growth, and the development of energy reserves in preparation for nursing and performing regular activities while carrying pregnancy's extra weight and volume. STUDY DESIGN, SIZE, DURATION: This study is based on a data set collected as part of a longitudinal, naturalistic investigation into the interactions between the stress (hypothalamic-pituitary-adrenal axis (HPAA)) and reproductive (hypothalamic-pituitary-gonadal axis (HPGA)) axes. Biomarkers of HPAA and HPGA function were quantified in first morning urinary specimens collected every other day from 22 healthy women who conceived a pregnancy during the study. We analyzed the longitudinal within- and between-individual variation in first morning urinary cortisol levels across the 12-week peri-conceptional period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were recruited from two rural, aboriginal, neighboring communities in Guatemala. Cortisol, estradiol and progesterone metabolites (estrone-3-glucuronide and pregnanediol glucuronide, respectively) and hCG levels were quantified in first morning urinary specimens using immunoassays to determine time of conception and confirm pregnancy maintenance. Linear mixed-effects models with regression splines were used to evaluate the magnitude and significance of changes in cortisol trajectories. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, maternal first morning urinary cortisol increased from 6 weeks prior to conception (geometric mean ± SD = 58.14 ± 36.00 ng/ml) to 6 weeks post-conception (89.29 ± 46.76 ng/ml). The magnitude of the increase between the pre- and post-conception periods varied significantly between women (likelihood ratio test statistic = 8.0017, P = 0.005). The peri-conceptional period is characterized by an increasing cortisol trajectory (+1.36% per day; P = 0.007) interrupted by a week-long decline immediately prior to conception (-4.02% per day; P = 0.0013). After conception cortisol increased again (+1.73% per day; P = 0.0008) for 4 weeks, fell in the fifth week (-6.60% per day; P = 0.0002) and increased again in post-conceptional week 6 (+8.86% per day; P = 0.002). Maternal urinary cortisol levels varied with sex of the gestating embryo. During gestational week 2, mothers carrying female embryos (N = 10) had higher mean cortisol levels than those carrying male embryos (N = 9) (t(17) = 2.28, P = 0.04). LIMITATIONS, REASONS FOR CAUTION: Our results are based on a relatively small sample (n = 22) of women. However, our repeated-measures design with an average of 27 ± 8 (mean ± SD) data points per woman strengthens the precision of estimates resulting in high statistical power. Additionally, our study population's high degree of ethnic and cultural homogeneity reduces the effects of confounders compared with those found in industrialized populations. This higher level of homogeneity also increases our statistical power. However, since there may be small differences in absolute cortisol values among ethnic groups, the social and biological background of our sample may affect the generalizability of our results. General patterns of HPAA activity, however, are expected to be universal across women. Finally, as there is, to the best of our knowledge, no evidence to the contrary, we assumed that urinary cortisol levels reflect HPAA activity and that changes in gonadal steroids across the menstrual cycle do not affect the levels of free cortisol measured in urine. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first longitudinal profile of basal maternal HPAA activity across the peri-conceptional period. A basic understanding of the normative (basal as opposed to stress-induced) changes in HPAA activity across this period is needed to accurately assess women's stress at this juncture. Importantly, changes in HPAA activity are likely to play a critical role in ovulation, fertilization, implantation, placentation and embryonic programing. Thus, this novel information should aid in the development of interventions aimed at preventing or moderating undesired effects of maternal physiological stress during the peri-conceptional period on reproductive outcomes as well as embryonic development. STUDY FUNDING/COMPETING INTERESTS: This research was funded by a CIHR IGH Open Operating grant (CIHR 106705) to P.A.N. and L.Z.; a Simon Fraser University (SFU) President's Start-up grant, a Community Trust Endowment Fund grant through SFU's Human Evolutionary Studies Program and a Michael Smith Foundation for Health Research Career Investigator Scholar Award to P.A.N.; an NSERC Discovery grant to L.Z.; a CIHR Post-Doctoral Fellowship to C.K.B. and an NSERC Undergraduate Student Research Award to H.M. and J.C.B. The funding agencies had no role in the design, analysis, interpretation or reporting of the findings. There are no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Measuring quality of care for rheumatic diseases using an electronic medical record.

OBJECTIVES: The objective of this study was twofold: (1) to determine how best to measure adherence with time-dependent quality indicators (QIs) related to laboratory monitoring, and (2) to assess the accuracy and efficiency of gathering QI adherence information from an electronic medical record (EMR). METHODS: A random sample of 100 patients were selected who had at least three visits with the diagnosis of rheumatoid arthritis (RA) at Brigham and Women's Hospital Arthritis Center in 2005. Using the EMR, it was determined whether patients had been prescribed a disease-modifying antirheumatic drug (DMARD) (QI #1) and if patients starting therapy received appropriate baseline laboratory testing (QI #2). For patients consistently prescribed a DMARD, adherence with follow-up testing (QI #3) was calculated using three different methods, the Calendar, Interval and Rolling Interval METHOD: . RESULTS: It was found that 97% of patients were prescribed a DMARD (QI #1) and baseline tests were completed in 50% of patients (QI #2). For follow-up testing (QI #3), mean adherence was 60% for the Calendar Method, 35% for the Interval Method, and 48% for the Rolling Interval Method. Using the Rolling Interval Method, adherence rates were similar across drug and laboratory testing type. CONCLUSIONS: Results for adherence with laboratory testing QIs for DMARD use differed depending on how the QIs were measured, suggesting that care must be taken in clearly defining methods. While EMRs will provide important opportunities for measuring adherence with QIs, they also present challenges that must be examined before widespread adoption of these data collection methods.

Polyamine conjugates of meso-tritolylporphyrin and protoporphyrin IX: potential agents for photodynamic therapy of cancers.

An efficient five-step synthesis method was developed to obtain tritolylporphyrin and protoporphyrin IX polyamine conjugates. These compounds were composed of either one polyamine unit (spermidine or spermine) covalently tethered to monocarboxyphenyl tritolylporphyrin or two molecules of polyamines borne by protoporphyrin IX. In each compound, an aliphatic spacer arm is linked to the N(4) polyamine position. Photocytotoxicity of these new compounds was evaluated against K562 human chronic myelogenous leukemia cells and compared to Photofrin II; protoporphyrin IX polyamine conjugates exhibited much stronger photocytocicity than Photofrin II and were shown to readily induce necrosis in treated cells.

A study of the stability of tri(glucosyloxyphenyl)chlorin, a sensitizer for photodynamic therapy, in human colon tumoural cells: a liquid chromatography and MALDI-TOF mass spectrometry analysis.

Asymmetrical glycoconjugated tetrapyrrolic macrocycles are under study as efficient sensitizers for photodynamic therapy (PDT). In this context, tri(meta-O-beta-glucopyranosyloxyphenyl)chlorin [TPC(m-O-Glu)(3)] 2a/3a was found to be four times more photoactive in vitro than Foscan. In a further study of this interesting glycoconjugate, its metabolism by cellular glycosidases in HT29 cells has to be explored. Cellular extracts of HT29 cells incubated with TPC(m-O-Glu)(3) (24h, 6microM) were analyzed by MALDI-TOF mass spectrometry and high performance liquid chromatography (HPLC). In MALDI-TOF mass spectra, the presence of compounds distinct from TPC(m-O-Glu)(3) (m/z 1151) were observed at m/z 989, 827 and 665 corresponding to the loss of one, two or three glucose units (162u) and were be ascribed to TPC(m-OH)(m-O-Glu)(2) 2/3b,b',b", TPC(m-OH)(2)(m-O-Glu) 2/3c,c',c" and TPC(m-OH)(3) isomers 2d/3d, respectively. The porphyrins resulting from chlorin oxidation TPP(m-O-Glu)(3) 4a, TPP(m-OH)(m-O-Glu)(2) 4b,b", TPP(m-OH)(2)(m-O-Glu) 4c,c" and TPP(m-OH)(3) 4d were also observed. The HPLC profile (lambda(anal)=420 nm) showed eight peaks consistent with mass spectra. The kinetics of deglucosylation was studied from HPLC profiles between 1 and 48h incubation. The concentration of triglucoconjugated and diglucoconjugated molecules was maximum around 3 and 8h incubation, respectively, whereas, totally deglucosylated species appeared only after incubation for more than 10h. The fully deglycosylated porphyrin TPP(m-OH)(3) is the final metabolite, being observed at a concentration 15 times higher than that of the remaining TPC(m-O-Glu)(3) 2a/3a. Compared to the photobiological activity of the parent molecule [TPC(m-O-Glu)(3)], a three times higher TPP(m-OH)(3) concentration was necessary to observe a similar in vitro photoactivity.

Synthesis and biological evaluation of thioglycosylated porphyrins for an application in photodynamic therapy.

The aim of this work is the synthesis of a new family of glycosylated porphyrins in which the sugar moieties are linked to the tetrapyrrole ring by a thioglycosidic bond. Two series have been designed. The first one corresponds to meso-aryl porphyrin derivatives. The second one has been obtained from protoporphyrin IX derivatization. Aryl-porphyrins were prepared from tristolyl o- and p-hydroxyporphyrins followed by bromoallylation and thioglycosylation with peracetylated S-glucose, mannose and galactose and deprotection. The other series has been synthesized from protoporphyrin IX dimethylester with a regioselective glycosylation of terminal alkenyl carbon. The UV-visible, NMR and MALDI mass spectra are presented. Photocytotoxicities of the synthesized compounds against K562 chronic leukaemia cell line has been evaluated.

Ultraviolet-B-induced inactivation of human OGG1, the repair enzyme for removal of 8-oxoguanine in DNA.

The OGG1 proteins are DNA N-glycosylases-apurinic-apyrimidinic lyases that are responsible for the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) base in DNA. The human enzyme (hOGG1) is a monomer of 345 amino acids containing 10 buried tryptophan (Trp) residues that are very sensitive to UVB irradiation. The photolysis quantum yield of these Trp residues is about 0.3 and 0.1 in argon- and air-saturated solutions, respectively. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry shows that several cleavage sites are identical under aerobic and anaerobic photolysis of Trp residues; one of them includes the active site. Western blots and polyacrylamide gel electrophoresis indicate that fragments of high molecular size are also formed. In addition to common photochemical paths with argon-saturated solutions, specific reactions occur in air-saturated solutions of hOGG1. The photolysis rate is inhibited by more than 50% on binding of hOGG1 to a 34mer oligonucleotide containing a single 8-oxoG-C base pair. Binding to the oligonucleotide with 8-oxoG-C induced a 20% quenching of the hOGG1 fluorescence, suggesting interaction of nucleic acid bases with the Trp residue(s) responsible for the photolysis. Using 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) and 8-oxoG as substrates, it is shown that protein photolysis induces photoinactivation of the DNA N-glycosylase activities. The excision of 8-oxoG is more affected than that of Me-FapyG at the same dose of UVB irradiation under both air and argon conditions. Besides the role of Trp residues, the possible involvement of Cys 253 in the photoinactivation process of hOGG1 is discussed.

Reaction of hen egg white lysozyme with Fischer-type metallocarbene complexes. Characterization of the conjugates and determination of the metal complex binding sites.

The introduction of heavy atoms into protein crystals is sometimes rendered difficult and tedious because of the poor specificity of the available reagents for particular target residues. On the other hand, transition organometallic chemistry offers an almost untouched field for this purpose. In particular, Fischer-type metallocarbene complexes of the general formula (CO)5W=C(OR1)R2 may be attractive reagents because they contain the heavy element tungsten and specifically target amino groups to form stable, covalent aminocarbene adducts. With a small protein such as hen egg white lysozyme (HEWL) with a limited number of potential binding sites, it was possible to form protein-aminocarbene conjugates that have an average of one aminocarbene moiety per protein molecule. RP-HPLC combined with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS analysis of the conjugates revealed that they were mixtures of the native protein, monoaminocarbenes and diaminocarbenes. Tryptic proteolysis experiments performed on the protein conjugates combined with MALDI-TOF-MS analysis of the aminocarbenic peptides allowed us to determine that lysines 13, 33, 97 and 116 were involved in the reaction of HEWL with (CO)5W=C(OMe)Me.

Gold nanoparticles containing redox-active supramolecular dendrons that recognize H2PO4-.

Gold nanoparticles have been functionalized with thiol dendrons containing three redox active amidoferrocenyl or silylferrocenyl units; using cyclic voltammetry, these dendronized gold nanoparticles recognize H2PO4-.

Fluorescence and mass spectrometry studies of meta-tetra(hydroxyphenyl)chlorin photoproducts.

The meta-tetra(hydroxyphenyl)chlorin (m-THPC), a second-generation sensitizer used in photodynamic therapy (PDT), is currently under clinical trial. In vivo fluorometry provides direct evidence that photobleaching processes are induced at the tumor site during PDT. Photoproduct formation has thus to be taken into account to fully understand PDT treatment. A preliminary step is to determine the fluorescence characteristics of photoproducts formed in solution. Solutions of m-THPC irradiated at 514 nm have been separated by HPLC using absorption and fluorescence detection. Six main photoproducts have been isolated. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) results, five fluorescent photoproducts emitting at 652 nm have been attributed to three mono-, one di- and one tri-hydroxy derivatives (m/z 697, 713 and 729, respectively). Fluorescence characteristics of mono-hydroxy forms were found to be similar to those of m-THPC, whereas fluorescence yields in di- and tri-hydroxy derivatives were very low. Another product, corresponding to a MALDI-TOF MS main signal at m/z 542, showed an absorption spectrum maximum at 522 nm while a weak fluorescence was detected at 480 nm. The loss of the Soret band suggests that this photoproduct results from the opening of the reduced pyrrole ring. The part played by each of these products in the photobleaching phenomenon of m-THPC is discussed.

Ultraviolet-B-induced damage to Escherichia coli Fpg protein.

We investigated the effect of UVB light (290 < or = lambda < or = 320 nm) on the structure and enzymatic activities of Escherichia coli Fpg protein (2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine-DNA glycosylase), a DNA repair enzyme containing a zinc finger motif and five chromophoric Trp residues. Irradiation with UVB light of air-saturated pH 7.4 buffered aqueous solutions of Fpg induces the formation of polymers as shown by sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis. In argon-saturated solutions, polymer formation produces a precipitate. The polymerization quantum yield is 0.07 +/- 0.01 and 0.15 +/- 0.02 in air- and argon-saturated solutions, respectively. In the polymerized Fpg protein, second-derivative absorption spectroscopy indicates that three and one Trp residues are destroyed in air- and argon-saturated solutions, respectively. Polymers are devoid of all three activities of the Fpg protein, whereas the unpolymerized protein retains full activities. Matrix-assisted laser desorption/ionization experiments demonstrate that polymer formation is accompanied by the formation of short polypeptides containing the first 32 or 33 residues of the N-terminal domain. Theses polypeptides are most probably formed by the photolytic cleavage of Fpg protein induced by light absorption by the adjacent Trp-34 residue.

Identification by mass spectroscopy of three major early proteins associated with virosomes in vaccinia virus-infected cells.

Virosomes are cytoplasmic sites of replication of vaccinia virus DNA and were prepared from virus-infected HeLa cells. The early virosomal proteins were 35S-labelled and SDS polyacrylamide gel electrophoresis revealed the presence of three major early 35S-labelled proteins of 34, 24 and 45 kDa. The masses of molecules present in the 34 and 24 kDa proteins were measured by the convenient and sensitive MALDI TOF mass spectroscopy technique. Identification of the three virosomal proteins was carried out by MALDI mass spectroscopy of corresponding tryptic digests. For each protein at least 13 measured masses matched, within less than 0.1 Da, calculated tryptic peptides of the vaccinia virus proteins H5R (34 kDa), E3L (24 kDa) and E5R (45 kDa). In addition, virosomes contained several structural proteins from the infecting virus and a 45 kDa keratin-related protein. This work demonstrates directly that the abundant early vaccinia virus proteins H5R, E3L and E5R are associated with the virosomes.

Coupling 2D SDS-PAGE with CNBr cleavage and MALDI-TOFMS: a strategy applied to the identification of proteins induced by a hypochlorous acid stress in Escherichia coli.

A protocol including 2D SDS-PAGE, electroblotting proteins onto nitrocellulose membranes, and CNBr cleavage, followed by MALDI-MS analysis of intact proteins and peptide fragments and a database search, has been optimized and applied to the rapid identification of the Escherichia coli response to hypochlorous acid. The methodology has proved to be efficient from the point of view of sensitivity (picomole range) and selectivity. In particular, MALDI analysis of proteins and CNBr fragments by directly dissolving the membrane in an acetone solution of matrix, without previous elution, is reliable and reproducible. The accuracy of the MW determination is somewhat reduced compared to that of methods involving elution and purification of proteins and digests; nevertheless, the utilization of large MW windows combined with the pI entry in database searches had allowed, for most of the spots, the selection of only one protein candidate. Finally, 19 proteins exhibiting a response to hypochlorous acid stress have been confirmed or identified on the basis of this protocol.

Kinetic analysis of the reaction between d(TTGGCCAA) and [Pt(NH3)3(H2O)]2+ by enzymatic degradation of the products and ESI and MALDI mass spectrometries.

The kinetics of the reaction between the octanucleotide d(TTGGCCAA) in the single-stranded form in pure water and the platinum complex [Pt(NH3)3(H2O)]2+ was investigated by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometries coupled with enzymatic degradation of the adducts. These methods led to the determination of specific rate constants of platination. The global rate constant characteristic of the formation of adducts on each 5'- or 3'-guanine were measured by electrospray ionization analysis. The ratios between the 5'- and 3'-adducts were determined from enzymatic degradation of the final reaction mixture and MALDI analysis. The platination in water is approximately eight times faster than in 0.1 M NaClO4. The selectivity of platination is a factor of 2 in favor of the 5'-guanine, and similar to that observed for the reaction between d(CTGGCTCA) and [Pt(NH3)3(H2O)]2+ in 0.1 M NaClO4.

Coupling of MALDI-TOF mass analysis to the separation of biotinylated peptides by magnetic streptavidin beads.

Extraction of biotinylated peptides by streptavidin magnetic beads has been directly coupled to the MALDI-TOF mass analysis. The elution of peptides from the beads is achieved by first mixing the beads with the MALDI matrix solution and removing, after a few minutes, the beads with a magnet; then, the matrix solution containing the biotinylated peptide is directly mass analyzed by MALDI. Three examples are presented to show the capabilities of this procedure to detect biotinylated peptides present at very low concentrations in complex mixtures. Detection limits of less than 100 finol can be achieved. Such a coupling strategy is of great interest to investigate peptide/ protein interactions.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of DNA-Pt(II) complexes.

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry has been used to characterize the reaction products of the 18-mer deoxyribonucleotide d(AACGGTTAACCGTTAATT) with [Pt(NH3)3(H2O)]2+ and cis-[Pt(NH3)2(H2O)2]2+. Characteristic peaks corresponding to different monofunctional adducts (18-mer+n[Pt(NH3)3]) (n = 1, 2, 3 and 4) have been observed with the triamino-monoaqua complex. With the diamino-diaqua cis-Pt complex, formation of a chelate (18-mer+[Pt(NH3)2]) involving two adjacent guanines has been demonstrated. A good correlation between MALDI and polyacrylamide gel electrophoresis results is observed.

2,4-diamino-7-hydroxy-pteridines, a new class of catabolites of methotrexate.

Methotrexate remains a commonly used drug in the chemotherapy of various malignancies. The known catabolites are 7-hydroxy-methotrexate, formed in the liver, and diamino-methyl-pteroic acid formed in the gut. We report for the first time evidence that 2,4-diamino-7-hydroxy-pteridine derivatives are present in the biological fluids of patients on high-dose methotrexate protocols. So far, two major derivatives have been identified as 2,4-diamino-6-hydroxymethyl-7-hydroxy-pteridine and 2,4-diamino-6-methyl-7-hydroxy-pteridine. In regard to the actual knowledge of the catabolism of pteridines, these compounds are presumably formed by intestinal bacteria during enterohepatic circulation of the drug. Their slow clearance from the body raises the question of possible interference of these compounds on pteridine-dependent enzymes, which might explain in part some of the toxic effects of methotrexate.

Maturation of caffeine N-demethylation in infancy: a study using the 13CO2 breath test.

Four premature neonates and eight infants 1-19 months old received caffeine for apnea. The usual morning oral dose was substituted by 1,3,7 13C-trimethylxanthine (13C-tri CAF) as the citrate salt. Five breath samples were collected the day before (day 1) and the day of 13C-tri CAF administration (day 2). Plasma (after each breath collection) and urine were collected on day 2. 13C-CO2 exhalation was determined by isotope ratio mass spectrometry. Caffeine and its metabolites were measured using high-pressure liquid chromatography. Assessment of the labeled CO2 in the breath revealed no detectable 13C-tri CAF N-demethylation activity in infants before 45 wk postconceptional age. However, demethylation (as urinary metabolites) has been detected before that age. Two-, 4-, and 6-h cumulative excretion of 13C-tri CAF as 13C-CO2 increased with postnatal age and correlated with caffeine plasma clearance (r = 0.840, p less than 0.01). These results were consistent with those obtained for urinary metabolites. In one infant (19 months old) the cumulative excretion of 13C-CO2 while crying was 65% of the value observed during quiet breathing. The measurement of caffeine demethylation using the caffeine CO2 breath test is feasible in infants and is a safe and noninvasive method to determine age related changes in P4501-dependent N-demethylase activity.