Extensive Interindividual Variation Research Articles

Higher than expected estradiol levels in aromatase inhibitor-treated, postmenopausal breast cancer patients

ABSTRACTObjective Vaginal estradiol is considered contraindicated in aromatase inhibitor (AI)-treated patients because of the risk of elevated estrogen levels. This leaves limited treatment options for patients experiencing gynecological symptoms. However, in clinical practice, no precise estimation has been performed of circulating estrogens and aromatase index in postmenopausal breast cancer patients on long-lasting AI or tamoxifen treatment.Methods Steroid hormones were measured using liquid chromatography tandem mass spectrometry (LC–MS/MS) and extraction radioimmunoassay (RIA). Postmenopausal AI-treated patients (n =33) were compared with tamoxifen-treated patients (n =34) and controls without vaginal treatment (n =56), with vaginal estradiol (n =25), or with estriol (n =11) treatment.Results By use of LC–MS/MS, median (range) estradiol plasma concentrations were 16.7 (2.4–162.6), 31.0 (13.4–77.1), 27.2 (7.8–115.8) and 33.3 (20.3–340.1) pmol/l in AI-treated breast cancer patients, tamoxifen-treated breast cancer patients, postmenopausal controls and postmenopausal controls on vaginal estradiol, respectively. The AI-treated group and subgroups had significantly lower estradiol and estrone concentrations than all other groups (p <0.05). There was extensive interindividual variation in estradiol concentration within the AI-treated group, measured using both LC–MS/MS (2.3–182.0 pmol/l) and extraction RIA (2.4–162.6 pmol/l). The AI-treated group had lower aromatase index compared to all other groups (p <0.05–0.001).Conclusion Circulating estrogen levels may have been underestimated in previous longitudinal studies of AI-treated breast cancer patients. Additional studies are required to further evaluate the role of circulating estrogens in breast cancer patients suffering from gynecological symptoms.

339 Sensitization to Contactants in Patients with Atopic Dermatitis

BackgroundAtopic dermatitis (AD) is a chronic inflammatory pruritic skin disease with extensive interindividual variation and multiple internal and external factors. In this study, we evaluated whether the atopic dermatitis severity (SCORAD index), gender, age, age onset or the presence of Allergic rhinitis (AR), Allergic conjunctivitis (AC) or Asthma has an influence on contact sensitization to common contactant allergens.Methods30 AD patients were evaluated in the Division of Allergy of Federal University of São Paulo. AD was diagnosed according to the Hanifin and Rajka's criteria and all patients were currently under regular treatment. Questionnaire (age, gender, age at onset, presence of AR, Asthma or AC), clinical examination and skin patch tests were carried out on all patients at the beginning of the study. Patients in regular use of oral CE; topical CE and/or calcineurin inihibitor use or having active AD lesions in the back were excluded from the study. Patch test was applied onto the upper back with 8 mm chambers attached with hypoallergenic tape and removed after 48 hours. The interpretation of the test reactions was performed at 48th and 96th hour.ResultsPositive Patch-test reaction occurred in 14/30 (46.6%). Among those with positive patch-test, Nickel was responsible for 42.8% and Thimerosal for 28.5%. All patients finished the study and no adverse reactions occurred. Positive and negative Patch-test groups found no statistically significant difference (P > 0.05) when comparing: SCORAD index, sex, age, age of onset and presence of AC, AR or asthma.ConclusionsAccording to our results, sensitization to common contact allergens in AD patients was more frequent than in normal subjects. Although we did not found an explanation to these findings, indiscriminate exposure to topic products should be avoided so that new sensitization or risk of deteriorating AD occurs. The benefits of avoidance to the contactants considered positive should be evaluated in the follow-up of these patients.

Liquid chromatography–tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

Thiopurines are S-substituted antimetabolites that are widely used in the treatment of hematological malignancies and as immunosuppressants. Because of extensive inter-individual variation in drug disposition and the significant toxicity associated with thiopurine therapy, there is a need for improved individualized treatment. We here present a fast and sensitive method for quantifying the pharmacological end-point of thiopurines, 6-thioguanine (TG) in chromosomal DNA. Purine nucleobases are released from DNA, etheno-derivatized with chloroacetaldehyde, separated by HILIC and quantified by tandem mass spectrometry using endogenous chromosomal guanine as internal standard. The method is linear up to at least 10 pmol TG/μg DNA and the limit of detection and quantification are 4.2 and 14.1 fmol TG/μg DNA, respectively. The matrix (DNA) had no effect upon quantification of TG. SPE recovery was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45–1190 fmol/μg DNA) with intra- and inter-day RSDs of less than 11%. The method uses 2 μg DNA/sample, which can easily be obtained from these patients.

Inter and Intra-Individual Variation of Factor VIII Half-Life In Severe Haemophilia A

AbstractAbstract 544 Introduction:Conventional dosing of FVIII prophylaxis in adults with hemophilia is based on 3 weekly or alternate day regimes of 20–40IU/kg, aiming for a trough level above 1IU/dL. However, pharmacokinetics of FVIII widely varies between individuals, thus the amount of factor concentrate required to maintain trough levels above 1IU/dL is also disparate. Mathematical modeling indicates that half-life has a significant influence over trough level. An individualized dose based on FVIII half-life may therefore allow for more appropriate dosing of prophylaxis in adults. For this approach to be effective, half-life results obtained from pharmacokinetic studies need to be consistent and representative of an individual's steady state. Although it is recognised that half-life increases from early childhood until adolescence, intra-individual variation of half-life in adults has not been examined. Hypotheses:We hypothesized that for patients >18years significant inter-individual variation, but minimal intra-individual variation, in half-life would be detected. Method:This single center study investigated inter- and intra-individual variation in FVIII half-life based on 140 pharmacokinetic studies conducted in 73 individuals with severe hemophilia A (FVIII:C < 1IU/dL). Data for all FVIII pharmacokinetic studies performed over a 5-year period were extracted from an in-house database. Patient demographics and laboratory results were verified against electronic patient records. All half-life studies were performed according to a set protocol and analyzed within the same clinical pathology accredited reference laboratory. Results were tested using the non-parametric Wilcoxin signed rank test. Data is presented as median and range and p-values < 0.001 were considered significant. Results:The median age of patients was 17.5 years (range 5.0– 54.0).The wide range in FVIII half-life data indicated substantial inter-individual variation within the complete population (median 9.9hrs, range 2.3–19hrs), replicated upon division of data into age specific categories (Fig. 1). [Display omitted] 36 patients had undergone multiple studies over the 5-year period. Of these, 21 had at least 3 data sets. FVIII half-life showed considerable intra-individual variation with 17 patients (81%) demonstrating a range of half-life greater than 2hrs.FVIII half-life data was normalized to nullify the effect of extensive inter-individual variation in half-life within the population. Figure 2 demonstrates mean difference in normalized FVIII half-life for all patients with multiple half-life results. Testing against a null hypothesis of no variation, we found significant intra-individual variation upon examination of the entire population (n= 36, p<0.0001). Following exclusion of data from children less than 6 years, significant intra-individual variation could still be detected for children 6–18 years (n=19, p< 0.0001), and adults (n= 16, p<0.0001). [Display omitted] Discussion:This study verifies expected inter-individual variation of pharmacokinetic profiles and appears to support the concept of individualized prophylaxis regimes in adults based on half-life data. However, considerable intra-individual variation in FVIII half-life in adult patients was registered. Mathematical models have indicated that even modest changes in half-life influence dosing requirements for FVIII prophylaxis. Intra-individual fluctuations in FVIII half-life will necessitate regular pharmacokinetic studies to ensure appropriate dosing. This will naturally affect the cost, utility and acceptability of treatment. Disclosures:No relevant conflicts of interest to declare.

Hepatic expression profiling in smolting and adult coho salmon ( Onchorhynchus kisutch)

Coho salmon are a critical Pacific salmon species that undergo complex physiological transformations as they migrate towards seawater and enter adult life stages. During these periods, coho may receive exposure to waterborne pollutants that coincide with outmigration through contaminated waterways and return to natal streams. However, little is known regarding the ontogenic modulation of gene expression during these critical life stages. Accordingly, the purpose of the present study was to characterize the hepatic transcriptome of smolting coho, adult males, and adult females by carrying out microarray analysis with a commercially available 16,000 cDNA element platform. Quantitative PCR (Q-PCR) analysis of genes involved in chemical biotransformation (cytochrome P450 isoforms 1A, and 2M1, glutathione S-transferase pi, microsomal GST), defense against metal exposure (metallothionein-A), and reproductive function (vitellogenin receptor) were developed for the purpose of analyzing specific genes of interest and to validate the microarray data. Microarray analysis identified 842 genes that were differentially expressed between smolts and adult males or females ( p<0.001 and more than 2-fold difference). These 842 genes were not differentially expressed between adult males and females and, therefore, can be interpreted as a smolt-specific transcriptional profile. Of these 842 genes, 275 were well annotated and formed the basis for further bioinformatics analysis. Many of the differentially expressed genes were involved in basic cellular processes related to protein biosynthesis and degradation (24%), ion transport (12%), transcription (8%), cell structure (8%) and cellular energetics (6%). The majority of differentially expressed genes involved in signal transduction and energy metabolism were expressed at higher levels in adult coho relative to smolts. However, genes associated with cellular protection against chemical injury (i.e. biotransformation, DNA damage repair, and protection against oxidative stress) did not generally differ among the groups. Q-PCR studies revealed extensive interindividual variation in mRNA expression, but were consistent with the microarray results ( R 2=0.74). Collectively, our results indicate differences in liver gene expression in young smolting coho salmon relative to adults and extensive interindividual variation in biotransformation gene expression. However, we did not find a global lack of hepatic biotransformation capacity or poor cellular detoxification response capacity in smolting cohos based on mRNA profiles.

Molecular properties and pharmacogenetics of a polymorphism of adenylyl cyclase type 9 in asthma: interaction between β-agonist and corticosteroid pathways

In asthma, the response to beta-agonists acting at beta2-adrenergic receptors (beta2AR) displays extensive interindividual variation. One effector for airway beta2AR, adenylyl cyclase type 9 (AC9), was considered a candidate locus for predicting beta-agonist efficacy in the absence and presence of corticosteroid treatment. One non-synonymous AC9 polymorphism has been identified, which results in substitution of Met for Ile at amino acid 772. Under standard culture conditions in stably transfected cells, we found decreased catalytic activity of Met772. However, cells cultured in the presence of glucocorticoid expressing Met772 had a significantly increased albuterol-stimulated adenylyl cyclase response (approximately 80%) when compared with those expressing Ile772 (approximately 20%, P=0.02). An equivalent increase in beta2AR expression was observed in both lines due to glucocorticoid, but AC9 expression was unaffected. The hypothesis that Met772-AC9 is associated with an improved albuterol bronchodilator response in asthmatics was investigated in 436 asthmatic children who were followed for 4 years and randomized to receive placebo or the inhaled corticosteroid budesonide. Met772 carriers on budesonide showed a significant improvement in forced expiratory volume in 1 s (P=0.005). Moreover, a highly significant interaction (P=0.002) was found for budesonide treatment and the AC9 polymorphism. These in vitro and human association studies are consistent with this AC9 polymorphism altering albuterol responsiveness in the context of concomitant inhaled corticosteroid administration, which is a common asthma regimen. The Met772-AC9 polymorphism represents one of most likely several multi-gene polymorphisms along the receptor-relaxation axis, which together may provide for a composite pharmacogenetic index for asthma therapy.

Variability in Aflatoxin B1–Macromolecular Binding and Relationship to Biotransformation Enzyme Expression in Human Prenatal and Adult Liver

Studies of transplacental transfer of aflatoxin B1 (AFB1) suggest that the developing human fetus may be a sensitive target for AFB1 injury. Because AFB1 requires metabolic activation to the reactive AFB1-8,9-exo-epoxide (AFBO) to exert its carcinogenic effects, ontogenic and interindividual differences in AFB1 biotransformation enzymes may underlie susceptibility to AFB1-induced cell injury. The present study was initiated to compare the rates of in vitro AFB1–DNA and AFB1–protein adduct formation among a panel of 10 adult and 10 second-trimester prenatal livers and to examine the relationship among AFB1 metabolizing enzyme expression and AFB1 binding. Mixtures of cytosolic and microsomal proteins from prenatal and adult livers catalyzed the formation of AFB1–DNA and AFB1–protein adducts at relatively similar rates, although greater individual variability in AFB1 adduct formation was observed in adult tissues. Extensive interindividual variation among adult tissues was observed in the expression of the AFB1 activation enzymes cytochrome P4501A2 (CYP1A2), CYP3A4/5, and lipoxygenase (LO). Prenatal CYP3A7 expression was also highly variable. LO expression was eightfold higher in prenatal liver tissues than adults, whereas the expression of the AFBO detoxification enzyme microsomal epoxide hydrolase was twofold higher in adult liver. The levels of the polymorphic glutathione S-transferase M1 (hGSTM1–1), which may potentially protect against AFBO injury, were higher in the hGSTM1–1-expressing tissues of adults in relation to prenatal livers. In general, there was not a strong relationship among AFB1–DNA or AFB1–protein adduct formation and expression levels of individual AFB1 metabolizing enzymes. In summary, despite the presence of marked individual and ontogenic differences in the expression of AFB1 metabolizing enzymes, human second trimester prenatal liver tissues compared to adults do not exhibit a marked sensitivity to the in vitro formation of macromolecular AFB1 adducts.

Interindividual Variation in Metabolism of Soy Isoflavones and Lignans: Influence of Habitual Diet on Equol Production by the Gut Microflora

The soy isoflavones, daidzein and genistein, and the lignans, matairesinol and secoisolariciresinol, are phytoestrogens metabolized extensively by the intestinal microflora. Considerable important evidence is already available that shows extensive interindividual variation in isoflavone metabolism, and we have investigated the extent of this variation in a crossover study of a soy-containing food low or high in isoflavones (each treatment period lasted for 17 days, and the 2 treatment periods were separated by a 25-day washout period) in 24 healthy subjects [19 women and 5 men, mean age 30 yr, range 19-40, mean body mass index 22.5 ±3.5 (SD) kg/m2]. There was a 16-fold variation in total isoflavonoid excretion in urine after the high-isoflavone treatment period. The variation in urinary equol excretion was greatest (664-fold), and subjects fell into two groups: poor equol excretors and good equol excretors (36%). A significant negative correlation was found between the proportion of energy from fat in the habitual diet and urinary equol excretion (r = -0.55; p = 0.012). Good equol excretors consumed less fat as percentage of energy than poor excretors (26 ± 2.3% compared with 35 ± 1.6%, p < 0.01) and more carbohydrate as percentage of energy than poor excretors (55 ± 2.9% compared with 47 ± 1.7%, p < 0.05). Interindividual variation in the urinary excretion of O-desmethylangolensin (O-DMA) was also apparent (76-fold after the high-isoflavone treatment period), but there was no relationship between equol excretion and O-DMA excretion. Enterolactone was the major lignan metabolite in urine and plasma but showed less interindividual variation than equol and O-DMA. It is suggested that the dietary fat intake decreases the capacity of gut microbial flora to synthesize equol.

Myoelectric activity during unilateral chewing in healthy subjects: Cycle duration and order of muscle activation

Statement of problem. Craniomandibular disorders, unilateral mastication, and asymmetry of masticatory muscles appear to be related to each other. Thus, it is of interest to investigate masticatory muscle activity during unilateral mastication in healthy subjects. Purpose. This study monitored contractile activity of the right and left masticatory muscles during right- and left-side gum chewing. Material and methods. Electromyographic techniques were used to determine chewing cycle duration and duration of contractile activity of the masticatory muscles (right and left masseter and anterior temporalis muscles) during unilateral chewing in 40 subjects without orofacial pain. The time-course of activation of the 4 muscles was also investigated. Results. Electromyographic traces showed extensive interindividual variation. In both right- and left-side chewing tests, and regardless of whether the masseter or the temporalis muscles were considered, mean duration of the contraction phase did not differ significantly between the working and nonworking sides. The working side temporalis contracted first, whether alone or at the same time as the other muscles. Conclusions. In healthy subjects, no significant differences in masticatory muscle activity should be expected between either the right and left or the working and nonworking sides. (J Prosthet Dent 1998;80:462-6.)

The natural history of multiple sclerosis: a geographically based study. 2. Predictive value of the early clinical course.

Controversy exists regarding the predictive value of the early clinical course of multiple sclerosis (MS). Three parameters often considered are the attack rate, the first interattack interval and the rate at which disability develops in the early years of the disease. We have recorded the time to reach successive levels of disability defined by the Kurtzke Disability Status Scale (DSS) in 1,099 MS patients followed at University Hospital, London, Canada between 1972 and 1984. Our population is particularly suitable because of its size, the high degree of ascertainment of cases in the community, and the regular follow-up provided. Life table analysis was used to compare survival in patients stratified according to the above three parameters using DSS 6 as end point. Significant differences were evident in the survival distributions. Despite the extensive interindividual variation in the rate at which disability developed, the early course of MS may be useful in determining the relative risk of rapid progression.

Enhancement of the bioavailability of propranolol and metoprolol by food.

The possible influence of food intake on the bioavailability of one nonselective and one cardioselective beta adrenoceptor antagonist, propranolol and metoprolol, was examined by serial determinations of the drug concentrations in blood of healthy subjects, taking single doses of the drugs both on an empty stomach and together with a standardized breakfast. The results indicate that food enhances the bioavailability of both propranolol and metoprolol. They also confirm that there is extensive interindividual variation in the bioavailability of these drugs.