Evaluation of a rapid micro-scale assay for tacrolimus by liquid chromatography-tandem mass spectrometry.

BACKGROUND: The immunosuppressive drug tacrolimus has complex and unpredictable pharmacokinetics, therefore regular monitoring is required in patients receiving tacrolimus therapy. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring tacrolimus concentrations in whole blood and have compared it with a microparticle enzyme immunoassay. METHODS: For the LC-MS/MS assay, samples were prepared in a 96-deep well microtitre plate by adding 10 micro L of blood to 40 micro L of 0.1 mol/L zinc sulphate solution. Proteins were precipitated by adding 100 micro L acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 20 micro L of the supernatant was injected into the LC-MS/MS system. A C18 cartridge (3 mm x 4 mm) was eluted with a step gradient of 50% to 100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid, at 0.6 mL/min. The column was maintained at 55 degrees C. RESULTS: The retention times were 0.98 min for ascomycin and 0.98 min for tacrolimus. Cycle time was 2.5 min, injection to injection. The analytes were monitored using a Quattro micro trade mark tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: m/z821 > 768 (tacrolimus) and m/z809 > 756 (ascomycin). The limit of quantitation was 0.5 micro g/L and the assay was linear up to 30 micro g/L. Precision of the method, over the concentration range 2.5-15.0 micro g/L, was < 7% within-batch and < 6% between-batch. Total time to analyse 24 samples including result generation was 90 min. CONCLUSION: We conclude that the LC-MS/MS method is quick, precise and robust and will provide a fast turn around of results for the transplant physician.

Height, societal threat, and the victory margin in presidential elections (1824-1992).

Based on the idea that height serves as a heuristic for judgments about status, dominance, and leadership potential, two hypotheses were tested: (1) Heights of U.S. presidential election winners are positively correlated with estimates of social, economic, and political threat in election years. (2) Height and victory margin are positively correlated regardless of the magnitude of estimates of social, economic, and political threat in election years. Both hypotheses were supported for the 43 elections from 1824 to 1992.

Birth order of past presidents and Schlesinger's history cycles: support for Stewart's leadership theory.

Stewart's hypothesis (1992) that adults who were only-children and first-born children are most suited to lead communities in more turbulent times while adults who were later-born children are most suited to lead in more tranquil times was tested by relating the male birth order of elected presidents to Schlesinger's 1986 public purpose and private interest periods. Consistent with Stewart's hypothesis, candidates who were only-children and first-born children tended to win during public purpose phases, which are characterized by the turbulence of greater political commitment, idealism, and broad social change.

The serotonin transporter gene and Parkinson's disease.

Dysfunction in the serotonin (5-hydroxytryptamine) system and reduced serotonin concentrations have been reported in patients with Parkinson's disease (PD). Serotonin concentrations in neural tissue are controlled by a presynaptic serotonin transporter protein that is encoded by a single gene. Therefore, we investigated whether a polymorphic region in the serotonin transporter gene is associated with PD. Three variable-number tandem repeat (VNTR) elements of the serotonin transporter gene were detected by polymerase chain reaction, those with 9, 10, 11 and 12 copies of the repeat element. The 10-copy VNTR element was significantly less common in patients with PD than controls in the univariate analysis (p < 0.05). Logistic regression analysis revealed no significant differences between patients (n = 198) and controls (n = 200) in the distribution frequencies of 9- and 12-copy alleles and combined genotypes (odds ratio = 1.20; p = 1.71). A positive family history of PD was a strong predictor of disease risk (odds ratio = 2.98; 95% confidence interval 1.51-5.87; p = 0.001). Although slight differences were observed between patient and control groups, these data suggest that defects in serotonin concentrations in patients with PD are unlikely to be due to polymorphisms in the serotonin transporter gene in this large Australian cohort; however, the inverse association observed with the 10-copy allele warrants further investigation.

Variations in the monoamine oxidase B (MAOB) gene are associated with Parkinson's disease.

The monoamine oxidase B gene (MAOB; Xp15.21-4) is a candidate gene for Parkinson's disease (PD) given its role in dopamine metabolism and its possible role in the activation of neurotoxins. The association of MAOB polymorphisms (a [GT] repeat allelic variation in intron 2 and an A-G transition in intron 13) with Parkinson's disease (PD) was studied in an Australian cohort of 204 (male:female ratio 1.60) people with PD and 285 (male:female ratio 1.64) age- and gender-matched control subjects. Genomic DNA was extracted from venous blood and polymerase chain reaction was used to amplify the appropriate regions of the MAOB gene. The length of each (GT) repeat sequence was determined by 5% polyacrylamide denaturing gel electrophoresis and a DNA fragment analyzer, while the G-A genotype was determined using 2% agarose gel electrophoresis. The G-A polymorphism showed no association with PD (odds ratio [OR] = 0.80; p = 0.51; 95% confidence interval [CI] = 0.42-1.53). There was a significant difference in allele frequencies of the (GT) repeat allelic variation between patients and control subjects (chi2 = 20.09; p<0.01). After statistical adjustment for potential confounders using a logistic regression analysis, the (GT) repeat alleles > or =188 base pairs in the intron 2 marker of the MAOB gene were significantly associated with PD (OR = 4.60; p<0.00005; 95% CI = 1.97-10.77). The 186 base pair allele was also significantly associated with PD (OR = 1.85; p = 0.048; 95% CI = 1.01-3.42). The GT repeat in intron 2 of the MAOB gene is a powerful marker for PD in this large Australian cohort.

The ACE deletion polymorphism is not associated with Parkinson's disease.

The deletion allele (D allele) polymorphism in the angiotensin converting enzyme (ACE) gene is associated with increased levels of the neuropeptide substance P in the basal ganglia and substantia nigra. A reduction of substance P levels in the brain occurs in Parkinson's disease (PD) and has been implicated in the pathogenesis of the disease. We investigated the hypothesis that the D allele may be protective towards PD by examining the frequency of the ACE (I/D) polymorphism in 178 PD cases (male:female ratio = 1.4) and 192 controls (male:female ratio = 1.5). ACE (I/D) genotype was determined using polymerase chain reaction and 3% agarose gel electrophoresis. Unadjusted chi-square analysis revealed no significant difference between genotype frequencies (chi2 = 3.30, p > 0.10) or allele frequencies (chi2 = 2.52, p > 0.10) between patient and control groups, although PD patients were less likely to be homozygous (OR = 0.80, 95% CI = 0.49-1.29) or heterozygous (OR = 0.80, 95% CI = 0.59-1.06) for the D allele. A stepwise logistic regression analysis of the ACE deletion and risk factor data confirmed that there was no significant association between the ACE deletion (D allele) polymorphism and PD (OR = 0.62, 95% CI = 0.35-1. 10, p = 0.10). This study does not support the hypothesis that the D allele of the ACE gene confers a protective effect with respect to PD.

No evidence of increased risk of colorectal cancer in individuals heterozygous for the Cys282Tyr haemochromatosis mutation.

BACKGROUND AND AIMS: Previous studies have suggested that increased body iron stores and heterozygosity for haemochromatosis are associated with an increased risk of colorectal carcinoma. The aim of this study is to determine if there is an association between (i) colorectal carcinoma and heterozygosity for the Cys282Tyr mutation of the haemochromatosis gene (HFE) and (ii) this mutation and tumour site or stage. METHODS: Two hundred and twenty-nine unselected patients (127 males, 102 females, mean age 68.0 years) with sporadic colorectal carcinoma and 228 controls (145 males, 83 females, mean age 69.7 years) were studied. DNA was tested for the presence of the Cys282Tyr mutation by digestion with Rsa1 and fragments separated by electrophoresis. RESULTS: Twenty-one patients with colorectal cancer and 23 control subjects were heterozygous for the Cys282Tyr mutation of HFE (relative risk 0.90). There was no association between heterozygosity of the Cys282Tyr mutation and tumour site or stage. CONCLUSIONS: Heterozygosity for the Cys282Tyr mutation of HFE does not appear to be a risk factor for colorectal carcinoma.

The epidemiology of Parkinson's disease in an Australian population.

A prevalence study of Parkinson's disease (PD) was conducted in the rural town of Nambour, Australia. There were 5 cases of PD in a study population of 1207, yielding a crude prevalence ratio of 414 per 100,000 (95% confidence interval; 53-775). We performed a separate case-control study involving 224 patients with PD and 310 controls from South East Queensland and Central West New South Wales, to determine which factors increase the risk for PD in Australia. A positive family history of PD was the strongest risk factor for the development of the disease (odds ratio = 3.4; p < 0.001). In addition, rural residency was a significant risk factor for PD (odds ratio = 1.8, p < 0.001). Hypertension, stroke and well water ingestion were inversely correlated with the development of PD. There was no significant difference between patients and controls for exposure to herbicides and pesticides, head injury, smoking or depression. The high prevalence of PD in Nambour may be explained by rural residency. However, the most significant risk factor for PD was a positive family hisotry. This demonstrates the need for improved understanding of the genetic nature of the disease.

Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state.

The heterogeneity of follicle stimulating hormone (FSH) and luteinizing hormone (LH) was investigated in five women aged 29.4 +/- 3.2 years (mean +/- SD) throughout their menstrual cycles and in five post-menopausal women aged 53.8 +/- 5.6 years. Chromatofocusing (pH range 7-4) revealed menstrual cycle stage- and postmenopausal-related differences in the serum gonadotrophin charge. There were differences in the proportion of FSH with an isoelectric point (pl) > 4.3 across phases of the menstrual cycle (P = 0.019): midcycle (MC) 50%; early to mid-follicular (EMF) 36%; late follicular (LF) 37%, luteal (L) 29% and following the menopause (PM) 17%. There was no significant difference in the proportion of LH with pl > 6.55 between midcycle (53%) and EMF, LF or L phases (36, 43 and 32% respectively); although all were greater than that found in the menopause (13%). Concanavalin A chromatography revealed less (P < 0.005) complex FSH and LH glycoforms at midcycle (63 and 13%) than in the EMF, LF and L phases (90 and 18; 90 and 20 and 93 and 24% respectively). Menopausal gonadotrophins were least complex (FSH 34%, LH 4%). There was a direct relationship between serum FSH and FSH pl/complexity, and less acidic FSH was associated with reduced FSH complexity. Increased oestradiol was associated with basic FSH isoforms during the menstrual cycle and reduced follicular phase FSH complexity. We conclude that changes in gonadotrophin glycoforms occur through the menstrual cycle which are related to changes in the prevailing steroid environment. Following the menopause oestrogenic loss resulted in acidic, relatively simple glycoforms.

The dopamine transporter gene and Parkinson's disease in a Chinese population.

We studied a variable number tandem repeat polymorphism within the dopamine transporter gene (DAT) for an association with Parkinson's disease in a Chinese population. Five alleles were detected, consisting of 6, 8, 9, 10, and 11 copies of the 40 base pair repeat sequence. The 10-copy allele was most common, accounting for 90% of alleles. There were no significant differences between the patients and the control subjects in the distribution frequencies of the alleles or genotypes. Therefore, this polymorphism is not associated with Parkinson's disease in Chinese populations.

Association of a polymorphism in the dopamine-transporter gene with Parkinson's disease.

The presynaptic dopamine transporter in nigral dopaminergic neurons confers susceptibility to the cytotoxic effects of the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Polymorphisms in the dopamine transporter might influence the susceptibility to such toxins. Therefore, we investigated whether a polymorphic region in the 3'-untranslated region of the dopamine-transporter gene is associated with idiopathic Parkinson's disease (PD). The frequency distribution of the alleles was significantly different between the patients (n = 100) and controls (n = 200, p < 0.05). The rare 11-copy allele was more common in the patients (odds ratio = 10.2, 95% confidence interval - 1.2-87.9, p < 0.025). The susceptibility of some people to PD may be conferred by polymorphisms in the dopamine-transporter gene that could lead to increased cellular accumulation of neurotoxic compounds in dopaminergic neurons.

The association between polymorphisms in the cytochrome P-450 2D6 gene and Parkinson's disease: a case-control study and meta-analysis.

We performed a case-control study to investigate the association of the poor metaboliser genotype of the cytochrome P450 2D6 gene with Parkinson's disease (PD). Genotyping was determined by the polymerase chain reaction followed by digestion with restriction enzymes. The poor metaboliser genotype was more frequent in 112 patients with PD than in 206 matched controls (odds ratio 1.7, 95% CI: 0.94-2.45). A meta-analysis of these results together with ten other published studies gave a pooled odds ratio for the poor metaboliser genotype of 1.47 (95% CI: 1.18-1.96, P=0.01). Thus, the poor metaboliser genotype has a small but highly significant association with PD which would be easily missed in small studies. Research now should focus on the mechanism of this association.

Sex differences, social desirability, masculinity, and the tendency to worry.

Relationships between worry and sex differences, social desirability, masculinity, and femininity were explored in this study. Data were obtained from 141 undergraduates who answered a questionnaire containing a worry scale, the Crowne-Marlowe (1964) Social Desirability Scale, the Bem (1974) Sex Role Inventory, the Trait Anxiety scale of the State-Trait Anxiety Inventory (Spielberger, Gorsuch, & Lushene, 1970), and several demographic items. Women reported significantly higher levels of worry than men did, and worry was significantly correlated with lower social desirability and with lower masculinity but not with femininity. However, multiple regression strategies revealed that sex differences in reported worry cannot be accounted for solely by variations in social desirability and masculinity. Also, sex differences in the tendency to worry were not eliminated by statistically controlling for trait anxiety, social desirability, and masculinity simultaneously.

Worry, anxiety, and preferred length of sleep.

Sixty-three female and 43 male undergraduates responded to a "worry" scale, a trait anxiety measure, and a question asking preferred length of sleep to clarify conflicting findings regarding the relationship between worry and anxiety and length of sleep (e.g., Hartmann, 1973; Hicks & Pellegrini, 1977; Kumar & Vaidya, 1984). Worry and length of sleep were positively correlated, supporting Hartmann's contentions. Anxiety was related to sleep in a U-shaped curvilinear fashion. Anxiety and worry were substantially correlated (r = .60) for subjects above the median on worry but were not correlated for subjects below the median. Worry may be the underlying construct in the positive relationship between anxiety and length of sleep for highly anxious persons. Comparison of high worry-low anxiety and low worry-high anxiety groups also suggests that our understanding of the relationships among sleep, anxiety, and worry may benefit from the simultaneous consideration of worry and anxiety factors.