Sex differences in cardiovascular responses to orthostatic challenge in healthy older persons: A pilot study.

BACKGROUND: Premenopausal women show a higher incidence of orthostatic hypotension than age-matched men, but there are limited data available on sex differences in cardiovascular responses to orthostatic challenge in healthy older persons. We investigated sex differences in hemodynamic and autonomic responses to orthostatic challenge in healthy older males and females. MATERIALS AND METHODS: Fourteen older healthy women and 10 age-matched men performed a sit-to-stand test (5 min of sitting followed by 5 min of standing). A Task Force® Monitor continuously measured the following beat-to-beat hemodynamic parameters: heart rate, systolic blood pressure, diastolic blood pressure, mean blood pressure, stroke index, cardiac index, and total peripheral resistance index. Cardiac autonomic activity, low-frequency (LF: 0.04-0.15 Hz) normalized (LFnuRRI) and high-frequency (HF: 0.15-0.4 Hz) normalized (HFnuRRI) components, and the ratio between LF and HF power (LF/HF) were calculated using power spectral analysis of heart rate variability. RESULTS: Across all hemodynamic parameters, there were no significant differences between the sexes at baseline and during standing. LFnuRRI (median: 70.2 vs. 52.3, p < 0.05) and LF/HF ratio (median: 2.4 vs. 1.1, p < 0.05) were significantly higher, whereas HFnuRRI (median: 29.8 vs. 47.7, p < 0.05) was lower among women at baseline. All other heart rate variability measures did not differ between the sexes. CONCLUSIONS: The data indicate that older women showed higher sympathetic and lower parasympathetic activity at rest compared to age-matched men. These results are contradictory to the observations from previous studies, which showed a reduced sympathetic and enhanced parasympathetic activity in women in all ages. Further studies are required to determine the underlying mechanisms contributing to higher incidence of orthostatic hypotension in older females.

Cryo-EM Structure Determination Using Segmented Helical Image Reconstruction.

Treating helices as single-particle-like segments followed by helical image reconstruction has become the method of choice for high-resolution structure determination of well-ordered helical viruses as well as flexible filaments. In this review, we will illustrate how the combination of latest hardware developments with optimized image processing routines have led to a series of near-atomic resolution structures of helical assemblies. Originally, the treatment of helices as a sequence of segments followed by Fourier-Bessel reconstruction revealed the potential to determine near-atomic resolution structures from helical specimens. In the meantime, real-space image processing of helices in a stack of single particles was developed and enabled the structure determination of specimens that resisted classical Fourier helical reconstruction and also facilitated high-resolution structure determination. Despite the progress in real-space analysis, the combination of Fourier and real-space processing is still commonly used to better estimate the symmetry parameters as the imposition of the correct helical symmetry is essential for high-resolution structure determination. Recent hardware advancement by the introduction of direct electron detectors has significantly enhanced the image quality and together with improved image processing procedures has made segmented helical reconstruction a very productive cryo-EM structure determination method.

Color-Doppler sonographic tissue perfusion measurements reveal significantly diminished renal cortical perfusion in kidneys with vesicoureteral reflux.

Vesicoureteral reflux (VUR) and its sequelae may lead to reduced renal perfusion and loss of renal function. Methods to describe and monitor tissue perfusion are needed. We investigated dynamic tissue perfusion measurement (DTPM) with the PixelFlux-software to measure microvascular changes in the renal cortex in 35 children with VUR and 28 healthy children. DTPM of defined horizontal slices of the renal cortex was carried out. A kidney was assigned to the "low grade reflux"-group if the reflux grade of the voiding cystourethrogram was 1 to 3 and to the "high grade reflux"-group if the reflux grade was 4 to 5. Kidneys with VUR showed a significantly reduced cortical perfusion. Compared to healthy kidneys, this decline reached in low and high grade refluxes within the proximal 50% of the cortex: 3% and 12 %, in the distal 50% of the cortex: 21% and 44 % and in the most distal 20 % of the cortex 41% and 44%. DTPM reveals a perfusion loss in kidneys depending on the degree of VUR, which is most pronounced in the peripheral cortex. Thus, DTPM offers the tool to evaluate microvascular perfusion, to help planning treatment decisions in children with VUR.

Role of variant Creutzfeldt-Jakob disease for safety of treatment with blood components: screening of lymphatic tissue is a potential tool for risk assessment.

BACKGROUND: Transmissible spongiform encephalopathies (TSEs) are neuropathological diseases caused by prions. Prions are infectious particles (PrPSc) which can induce bovine spongiform encephalopathy and most likely also the related infectious disease, variant of Creutzfeldt-Jakob disease (vCJD). The exposure of humans to orally ingested BSE agent in contaminated meat products presumably led to the emergence of vCJD. In vCJD, prions can be detected immunohistochemically not only in neuronal tissue but also in lymphoreticular tissue. vCJD is of significance in transfusion medicine because of the hypothetical transmission of prions by blood products. METHODS: An immunohistochemistry method was used to allow screening for vCJD in human lymphoreticular tissue. RESULTS: PrPSc can be detected in the cerebrum and cerebellum of patients with sporadic Creutzfeldt-Jakob disease (sCJD) and in the lymph nodes, tonsils, and spleen of vCJD patients. This method has the major advantage of working in fixed specimens which are routinely saved in departments of pathology and therefore allows screening of large numbers of archived human lymphoreticular tissues in different regional areas and from different time points. Scrapie-positive lymphoreticular sheep tissue reacts similarly to human tissue of vCJD affected patients and is available in sufficient amounts to be used as positive control in screening programs. CONCLUSION: A method is provided which is a feasible tool for an epidemiological screening program to assess the prevalence of the assumed infectious agent of vCJD, PrPSc, in various populations.

IgA and IgG autoantibodies against alpha-fodrin as markers for Sjögren's syndrome. Systemic lupus erythematosus.

OBJECTIVE: To determine the prevalence of IgA and IgG autoantibodies against alpha-fodrin in patients with primary and secondary Sjögren's syndrome (SS) and controls. METHODS: An ELISA detecting IgA and IgG antibodies against alpha-fodrin was developed. We examined the prevalence of IgA and IgG antibodies against alpha-fodrin in patients with primary and secondary SS, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) and blood donors. RESULTS: IgA antibodies against alpha-fodrin were detected in 64% of patients with primary SS (n = 85), 47% of patients with secondary SS and SLE (n = 15), and 86% of patients with secondary SS and RA (n = 7). IgA autoantibodies against alpha-fodrin were detected in only one of 160 sera obtained from blood donors and in one of 50 and 2 of 12 sera obtained from SLE and RA patients without sicca syndrome, respectively. The prevalence of IgG antibodies against alpha-fodrin in SS was lower: they were detected in 55% of sera obtained from patients with primary SS, 40% of patients with secondary SS and SLE, and in 43% of patients with secondary SS and RA. Three of 160 sera from blood donors and one of 50 and 5 of 12 sera from SLE and RA patients without sicca syndrome, respectively, contained IgG antibodies against alpha-fodrin. CONCLUSION: IgA rather than IgG antibodies against alpha-fodrin are specific for and frequently observed in primary and secondary SS and are useful markers for this autoimmune disorder.

Long-term therapeutic drug monitoring of clozapine and metabolites in psychiatric in- and outpatients.

RATIONALE: Clozapine is a unique antipsychotic drug, outstanding for its lack of extrapyramidal side-effects and its superior efficacy in refractory schizophrenia. However, an unambiguous concentration-response relationship has not yet been established. OBJECTIVE: We investigated serum concentrations of clozapine, norclozapine and clozapine-N-oxide in psychiatric in- and outpatients to identify particular metabolic patterns in clozapine responders and non-responders and putative threshold levels for clozapine response. METHODS: Psychiatric assessments, CYP2D6 genotype, and weekly serum concentrations of clozapine, norclozapine and clozapine-N-oxide were obtained in 34 adult schizophrenic in-and outpatients (18 men, 16 women) during 10 weeks of clozapine treatment with a naturalistic dose design. RESULTS: Responders (n=21) displayed significantly lower serum concentrations of clozapine corrected for dose compared to non-responders (n=13; P<0.05), while none of the other parameters (absolute clozapine concentration, metabolite ratios, gender) were different. Smokers had significantly lower dose-corrected clozapine concentrations. A positive correlation was observed between age and average steady state clozapine concentrations. CONCLUSIONS: These findings indicate a possible link between CYP activity and response to clozapine that is not mediated through differences in serum concentrations. No clinically meaningful pattern in serum parameters could be identified that differentiates responders from non-responders. Thus, clozapine TDM seems ineffective for predicting clinical response. Smoking behavior is a major determinant of clozapine clearance while CYP2D6 genotype does not impact clozapine disposition.

Rheumatoid factors in systemic lupus erythematosus: association with clinical and laboratory parameters. SLE study group.

The prevalence and clinical and laboratory associations of IgM, IgG and IgA rheumatoid factors (RF) were determined in 352 patients with systemic lupus erythematosus (SLE). IgM, IgG, and IgA class RF were detected in 17.9%, 20.5%, and 20.5% of the sera, respectively. RF were associated with sicca syndrome, hypergammaglobulinemia, high titer of antinuclear antibodies, anemia, SSA- and SSB-antibodies, and with the presence of HLA-DR3. RF correlated negatively with nephritis and livedo racemosa. Moreover, we observed an association of RF and parameters of inflammatory activity such as elevated erythrocyte sedimentation rate (ESR) and leukopenia. Analysis of immunoglobulin classes revealed that laboratory parameters of inflammatory activity, SSA- and SSB-antibodies and HLA-DR3 correlated with IgA RF only. IgA RF define a subgroup of SLE patients characterized by distinct autoimmune phenomena and high disease activity in the absence of nephritis.

Neutrophil intracellular pH and phagocytosis after thermal trauma.

Severe burn trauma induces an acquired dysfunction of neutrophil granulocytes. As neutrophil function is considerably influenced by intracellular pH (pH(i)), the pH(i) of blood neutrophils was longitudinally determined in 19 patients with major burns. pH(i) was measured by a flow cytometric method using the pH-sensitive fluoroprobe carboxy-semi-naphthorhodafluor-1; mechanisms influencing the pH(i) were examined by addition of amiloride (inhibition of Na(+)/H(+) countertransport), diphenylene iodonium (inhibition of NADPH oxidase) and N-formyl-methionyl-leucyl-phenylalanine (activation of H(+) extrusion). The neutrophil phagocytic activity was measured in parallel. Patients showed distinct alterations of neutrophil pH(i), depending on whether they developed sepsis in the postburn period or not. In the sepsis patients pH(i) did not deviate from the values found in healthy volunteers in the first days after injury, but rose afterwards, with significant intracellular alkalinization in the second postburn week (P<0.05). In contrast, patients without sepsis had increased pH(i) in the first (P<0.01 at days 1-2), but not in the second week after burn trauma. Inhibition studies showed that postburn intracellular alkalinization is not solely caused by activation of Na(+)/H(+) countertransport. A clear relation between pH(i) changes and phagocytosis could not be established.

[Gene therapy possibilities in plastic surgery]. / Gentherapeutische Möglichkeiten in der plastischen Chirurgie.

Advances in gene technologies have meanwhile reached plastic surgery. Important contributions in this field (which are not all included in the paper) come not only from plastic surgeons, but also from neighboring specialities like dermatology, trauma surgery, orthopedics and vascular surgery. The uniting principle for all this work is improving wound healing and reconstructing tissue defects taking into consideration functional and aesthetic aspects. Gene-therapy is gaining further importance in the clinical field of plastic surgery. In this regard, every clinician has to be aware of the fact that progress in experimental and experimental-clinical work will be achieved only with the help of basic science. On the other hand, basic science needs the clinical input to get relevant patient-oriented studies started. Further intensive cooperation between clinicians and basic scientists is therefore mandatory. In plastic surgery, 2 years ago we founded a forum called ECSAPS (European Conference of Scientists and Plastic Surgeons), which takes place in European city every year.

CYP2D6 genotyping as an alternative to phenotyping for determination of metabolic status in a clinical trial setting.

The emerging application of pharmacogenomics in the clinical trial setting requires careful comparison with more traditional phenotyping methodologies, particularly in the drug metabolism area where phenotyping is used extensively. The research objectives of this study were 1) to assess the utility of cytochrome P450 2D6 (CYP2D6) genotyping as an alternative to traditional phenotyping as a predictor of poor metabolizer status; 2) to identify issues for consideration when implementing CYP2D6 genotyping in clinical trials; and 3) to outline the advantages and disadvantages of CYP2D6 genotyping compared with phenotyping. DNA samples obtained from 558 previously phenotyped individuals were blindly genotyped at the CYP2D6 locus, and the genotype-phenotype correlation was then determined. The CYP2D6 genotyping methodology successfully predicted all but 1 of the 46 poor metabolizer subjects, and it was determined that this 1 individual had a novel (presumably inactive) mutation within the coding region. In addition, we identified 2 subjects with CYP2D6 genotypes indicative of poor metabolizers who had extensive metabolizer phenotypes as determined by dextromethorphan/dextrorphan ratios. This finding suggests that traditional phenotyping methods do not always offer 100% specificity. Our results suggest that CYP2D6 genotyping is a valid alternative to traditional phenotyping in a clinical trial setting, and in some cases may be better. We also discuss some of the issues and considerations related to the use of genotyping in clinical trials and medical practice.

Clozapine-induced agranulocytosis and hereditary polymorphisms of clozapine metabolizing enzymes: no association with myeloperoxidase and cytochrome P4502D6.

The pathomechanisms of most drug-induced agranulocytoses are unclear; however, there are some studies pointing to genetic determinants. Some drug-induced agranulocytoses such as clozapine-induced agranulocytosis (CA) may be regarded as an idiosyncratic drug reaction because of its preclinical and clinical characteristics. To study some aspects of the genetic background of CA further, polymorphisms of specific metabolizing enzyme systems of clozapine were examined. Thirty-one schizophrenic patients with CA and 77 schizophrenic comparison subjects without this adverse effect underwent genotyping of a recently discovered G(-463)A polymorphism of myeloperoxidase (MPO) gene and cytochrome P4502D6. Neither the MPO mutation nor specific genotypes of cytochrome P4502D6 were associated with CA. Both were equally distributed among CA patients and controls. Thus, our data suggest lack of evidence of an association of CA and genetically variable activity of these specific drug metabolizing enzymes; however, this may be due to statistical reasons only. Thus, further studies with greater CA samples are necessary to draw final conclusions about these genetically based hypotheses.

Flavin monooxygenase 3 (FMO3) polymorphism in a white population: allele frequencies, mutation linkage, and functional effects on clozapine and caffeine metabolism.

AIM: The flavin-containing monooxygenase 3 (FMO3) has been shown to be genetically polymorphic. In vitro, the enzyme contributes to the N-oxidation of clozapine, caffeine, and several other drugs. We therefore wanted to analyze population frequencies and allelic linkage of FMO3 mutations and their functional effect on the metabolism of clozapine and caffeine. METHODS: This study included 204 patients treated with clozapine for schizophrenia and 192 healthy volunteers receiving a 100 mg oral test dose of caffeine. FMO3 polymorphisms M66I, P153L, E158K, V257M, E305X, E308G, and R492W were analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Ratios of serum clozapine N-oxide over clozapine and of urine theobromine versus paraxanthine were used as in vivo indicators of FMO3 activity. RESULTS: From the known FMO3 amino acid variants, only K158 (frequency 0.426), G308 (0.225), and M257 (0.069) were found; mutations I66, L153, X305, and W492 were not found in the 396 subjects. Linkage analysis revealed seven different alleles; the most frequent of these was the wild-type E158-V257-E308 (0.534), followed by K158-V257-G308 (0.199) and K158-V257-E308 (0.192). Subjects with these frequent variants of FMO3, however, did not differ in clozapine N-oxidation or caffeine oxidation compared with the wild-type. CONCLUSION: There are several genetic polymorphisms for the FMO3 enzyme. The effects on the metabolism of caffeine or clozapine could not be shown, indicating that the mutations have only minor functional effects or that substrate affinity is too low to be clinically relevant.

Failure to respond to treatment with typical antipsychotics is not associated with CYP2D6 ultrarapid hydroxylation.

AIMS: To investigate whether or not there is a correlation between failure to respond to typical antipsychotics and CYP2D6 ultrarapid metaboliser status. METHODS: CYP2D6 phenotype (metaboliser status) was assigned following genotyping for gene duplication, as well as for the CYP2D6*3, CYP2D6*4, and CYP2D6*5 null alleles in 235 treatment-refractory patients and 73 nonrefractory patients. RESULTS: Four (1.7%) of the 235 treatment-refractory subjects were positive on the duplication assay, but, of these, two were found to represent duplications of a null allele (CYP2D6*4 ), therefore leaving only two (0.85%) positive for duplication of a wild type allele (ultrarapid metabolisers). Three (4.1%) of the nonrefractory subjects had a genotype consistent with ultrarapid metaboliser status. Fisher's exact test gave a two-tailed P value of 0.091, i.e. a trend towards an excess of ultrarapid metabolisers in the nonrefractory group, which was in the opposite direction to that predicted by our hypothesis. CONCLUSIONS: Although the results show a trend towards an excess of ultrarapid metabolisers in the nonrefractory group, the percentages in the two groups of patients are both within the range for ultrarapid metabolisers in Caucasian populations. Our data are not consistent with ultrarapid metaboliser status being a major cause of failure to respond to typical antipsychotics.

Frequency of cytochrome P450 CYP2C9 variants in a Turkish population and functional relevance for phenytoin.

AIMS: The genetically polymorphic cytochrome P450 enzyme CYP2C9 metabolizes many important drugs. We studied the frequency of the amino acid variants cysteine144 (CYP2C9*2 ) and leucine359 (CYP2C9*3 ) in a Turkish population and the correlation between genotype and phenotype using phenytoin as probe drug. METHODS: CYP2C9 alleles *2 and *3 were measured in 499 unrelated Turkish subjects by PCR and restriction fragment length pattern analysis. Phenotyping was performed in a subgroup of 101 volunteers with a single oral dose of 300 mg phenytoin and concentration analysis in serum drawn 12 h after dosage. RESULTS: CYP2C9 allele frequencies in 499 unrelated Turkish subjects were 0.794 for CYP2C9*1, 0.106 for CYP2C9*2 and 0. 100 for CYP2C9*3. Mean phenytoin serum concentrations at 12 h after dosage were 4.16 mg l-1 (95% CI 3.86-4.46) in carriers of the genotype CYP2C9*1/1, 5.52 mg l-1 (4.66-6.39) in CYP2C9*1/2, and 5.65 mg l-1 (4.86-6.43) in CYP2C9*1/3. These differences were significant and accounted for 31% of total variability in phenytoin trough levels. Mean 12 h concentration ratios of 5-(para-hydroxyphenyl)-5-phenylhydantoin/phenytoin (p-HPPH/P) were 0. 43 (0.39-0.47) for CYP2C9*1/1 compared with 0.26 (0.21-0.31) for CYP2C9*1/2, 0.14 (0.13-0.14) for CYP2C9*2/2, 0.21 (0.18-0.24) for CYP2C9*1/3, and 0.02 for CYP2C9*3/3; all mutant genotypes were significantly different compared with CYP2C9*1/1. CONCLUSIONS: Frequency of the two CYP2C9 variants in Turkish subjects was in a similar range as in other Caucasian populations. A significant proportion of the interindividual variability in phenytoin trough levels is explained by the genotypes. The 12 h serum concentrations after a single phenytoin dose may be used for routine phenotyping of CYP2C9 mediated metabolic clearance and the p-HPPH/P ratios may be even more sensitive indicators of CYP2C9 activity.

Procalcitonin as a marker for the early diagnosis of severe infection after thermal injury.

High serum concentrations of procalcitonin (PCT), the 116 amino acid precursor protein of the hormone calcitonin, have been found in patients with various bacterial infections, particularly in those with sepsis. Because recent reports have shown that serum PCT constitutes a useful parameter for the diagnosis of sepsis in patients with several clinical conditions, a temporal analysis of the PCT concentrations in the plasma of 19 patients with severe burns (median body surface area burned, 32%) was conducted retrospectively. Nine patients were classified as septic on the basis of standardized clinical and laboratory parameters. Compared with the nonseptic group, these patients showed higher plasma PCT throughout the study period (median concentrations of septic vs nonseptic patient groups: 0.4 vs. 0.2 microg/L on postburn day 2; 1.0 vs. 0.3 microg/L on postburn day 4; 5.5 vs. 0.3 microg/L on postburn day 7; 10.8 vs. 0.5 microg/L on postburn day 9; 4.2 vs. 0.4 microg/L on postburn day 12; and 1.7 vs. 0.5 microg/L on postburn day 14), with differences considered to be significant (P<.05) from day 7 on. In contrast, differences in the plasma C-reactive protein concentrations were less pronounced and never reached statistical significance. PCT concentrations exceeding 15 microg/L were only observed in the 3 patients who died of sepsis-induced multiple organ failure. In addition to absolute PCT, individual time courses were also of diagnostic value. PCT is a highly efficient laboratory parameter for the diagnosis of severe infectious complications after a burn injury.

Low frequency of defective alleles of cytochrome P450 enzymes 2C19 and 2D6 in the Turkish population.

BACKGROUND AND OBJECTIVES: The genetically polymorphic cytochrome P450 enzymes 2Cl9 (CYP2Cl9) and 2D6 (CYP2D6) contribute to the metabolism of about 30% of all drugs. For analysis of the ethnic-related differences in drug disposition and as a preparation for routine genotyping, we examined CYP2C19 and CYP2D6 mutations in a large Turkish population. METHODS: CYP2C19 and CYP2D6 alleles were determined with use of genomic deoxyribonucleic acid from 404 unrelated Turkish individuals. CYP2C19 alleles *1 to *5 and CYP2D6 alleles *1 to *12, and *14, *15, and *17 were measured by polymerase chain reaction-restriction fragment length polymorphism assays. RESULTS: From 404 subjects genotyped for CYP2C19, allele frequencies of CYP2C19*1 (wt), CYP2C19*2 (ml), and CYP2C19*3 (m2) were 0.88, 0.12, and 0.004, respectively; mutations m3 and m4 were not found. Four individuals (1.0%) were predicted to be poor metabolizers (CYP2C19*2/*2), a significantly lower frequency compared to Middle European populations. Among 404 subjects genotyped for CYP2D6, most frequent alleles were CYP2D6*1 (allele frequency 0.37), *2 (0.35), *4 (0.11), *10 (0.06), duplications *1x2, *2x2, or *4x2 (0.06), *5 (0.01), and *17(0.01). Overall, six subjects (1.49%) were predicted to be CYP2D6 poor metabolizers, and 35 subjects (8.66%) were predicted to be ultrarapid metabolizers as a result of CYP2D6 gene duplications. CONCLUSION: Obviously, within Europe there is a north-south gradient, with decreasing frequency of poor metabolizers of CYP2C19 and CYP2D6 to the south and a corresponding increase of ultrarapid metabolizers of CYP2D6. As in other white groups, only CYP2C19*2 plays a relevant role for the CYP2C19 poor metabolizer phenotype. The mutational spectrum of CYP2D6 indicated partial ethnic relationships to Asian and African populations.

Association between reduced human leukocyte antigen (HLA)-DR expression on blood monocytes and increased plasma level of interleukin-10 in patients with severe burns.

Severe thermal injury causes an immune dysfunction which includes a decrease of monocyte human leukocyte antigen DR (HLA-DR) expression. Interleukin-10 exerts a negative influence on this parameter in vitro. In this study we determined the prognostic value of reduced monocyte HLA-DR expression with regard to infectious complications, and the in vivo association between monocyte HLA-DR and plasma interleukin-10 concentration. Both quantities were measured serially in 19 patients with severe burns. HLA-DR expression was determined by direct immunofluorescence on a flow cytometer, and interleukin-10 was measured by ELISA. After burn trauma the percentage of HLA-DR expressing monocytes fell markedly (median: 53% at day 2, 36% at day 4, 31% at day 7, 28% at day 9, 35% at day 12, and 42% at day 14; compared to 93% for healthy volunteers). Moreover, patients who became septic showed lower monocyte HLA-DR expression than non-septic patients; the differences were significant at day 2 (p < 0.01) and day 7 (p < 0.05). Plasma concentrations of interleukin-10 increased after thermal injury (median: 40 ng/l at day 2, 43 ng/l at day 4, 77 ng/l at day 7, 120 ng/l at day 9, 63 ng/l at day 12, and 82 ng/l at day 14). Individual HLA-DR expression and interleukin-10 concentration were negatively correlated, the association reaching statistical significance at day 4 (p=0.006) and day 7 (p=0.031). Thus, after severe burn injury monocyte HLA-DR expression has prognostic value and is negatively associated with interleukin-10 plasma concentration.

Functional significance of a C-->A polymorphism in intron 1 of the cytochrome P450 CYP1A2 gene tested with caffeine.

AIMS: The cytochrome P450 enzyme CYP1A2 metabolises several drugs and carcinogens. We wanted to determine how much of the variability of CYP1A2 activity is explained by a newly discovered gene polymorphism in intron 1. METHODS: A single nucleotide polymorphism in intron 1 of the CYP1A2 gene at position 734 downstream of the first transcribed nucleotide was identified by DNA sequence analysis. The functional significance of this C/A polymorphism was assessed in 185 healthy Caucasian non-smokers and in 51 smokers by genotyping and phenotyping using caffeine (100 mg oral dose). RESULTS: Out of the total sample, 46% were homozygous for the variant A, 44% were heterozygous, and 10% were homozygous for the variant C. The ratio of 1,7-dimethylxanthine (17X) plus 1,7-dimethyluric acid divided by caffeine in 0-5 h urine samples from 185 non-smokers did not differ significantly between the three CYP1A2 genotypes. In the 51 smokers, analysis of variance revealed significant differences in the 5 h plasma 17X/caffeine ratios between the genotypes (P=0.008, F-test). The mean ratio was 1.37 in carriers of the A/A genotype, 0.88 in heterozygotes and 0.82 in carriers of C/C. The mean difference between the A/A and C/A groups was 0.48 (95% confidence interval 0. 15-0.81; P=0.01). CONCLUSIONS: The A/A genotype, which may represent a CYP1A2 high inducibility genotype, may either be a direct cause of increased CYP1A2 activity, or be genetically linked to polymorphisms conferring high inducibility. Further studies are needed to define the role of this polymorphism on the pharmacokinetics of drugs metabolised by CYP1A2 and in the activation of carcinogens.

Detection of the GSTM1*0 allele by long polymerase chain reaction.

Approximately 50% of individuals in most human populations completely lack activity of the detoxifying enzyme glutathione S-transferase M1. The medical consequences of this deficiency have been extensively investigated in molecular epidemiological studies, but possible differences of the highly active homozygous genotype versus the moderately active heterozygous genotype could not be considered because many currently used polymerase chain reaction (PCR) assays cannot distinguish the homozygous genotypes GSTM1 *A/A and GSTM1*B/B from the heterozygous genotypes GSTM1*A/0 and GSTM1*B/0. Here, we describe that long PCR is suitable for this purpose by specifically producing a signal for the GSTM1*0 allele. Based on the published cluster of GSTM genes (GSTM1 to -M5), a 13-kb segment spanning the site of the GSTM1 deletion was amplified using a GSTM2-specific forward primer (5'-CATCGCTTATGATGTCCTTGAGAGAAACCAAG-3') and a reverse primer, which is specific for the upstream region of GSTMS (5'-GCGTTTCTGAGGACTGGACTGATGATCG-3'). Any failure in the amplification was controlled by co-amplification of a 15-kb human tissue plasminogen activator gene fragment. The GSTM1*0-specific 13-kb amplicon appears in all carriers of one or two null alleles, but in none of the 14 tested GST1*A/B samples which served as controls since individuals with this genotype are known to lack any GSTM1*0 allele. While conventional PCR assays for detection of the GSTM1 deletion differentiated homozygously deficient from hetero- or homozygously active individuals, this long PCR assay differentiates homozygously active from hetero- or homozygously deficient individuals. Using both assays, an unambiguous differentiation into carriers of zero, one or two active alleles of GSTM1 is possible.