[New insight in therapeutic anticoagulation by Coumarin derivatives]. / Neue Einsichten in die orale Antikoagulations-therapie mit Cumarinen.

The recent identification of vitamin K epoxid-reductase complex (VKORC1) contributed significantly to our mechanistic understanding of the vitamin K cycle. VKORC1 protein is targeted by Coumarins. Its enzymatic activity represents the rate-limiting step in the vitamin K cycle and gamma-carboxylation of vitamin K dependent proteins. Possibly, VKORC1 is the only component of VKOR activity. Mutations as well as polymorphisms in coding and non-coding regions of the VKORC1 gene have been shown to cause both partial to total coumarin resistance and coumarin sensitivity. Availability of molecular diagnostics (VKORC1, CYP2C9) and laboratory analysis by HPLC (determination of coumarin, vitamin K and vitamin K epoxide levels) is helpful in detection of hereditary and acquired factors influencing coumarin therapy. In the future, these tools might lead to an individualized and safer oral anticoagulation therapy. Furthermore, daily low-dose vitamin K supplementation may improve stability of coumarin-based anticoagulation. In the perspective of the coming new oral anticoagulants, the efficacy and safety profile of the "old" anticoagulants is of major importance. The well established and oeconomic coumarin drugs will benefit from a pharmacogenetic and nutritive adjusted optimization of therapy.

VKORC1: molecular target of coumarins.

The genetic diagnosis of a single family with combined vitamin K-dependent clotting factor deficiency (VKCFD2, OMIM #607473) finally led to the identification and molecular characterization of vitamin K epoxide reductase (VKORC1). VKORC1 is the key enzyme of the vitamin K cycle and the molecular target of coumarins, which represent the most commonly prescribed drugs for therapy and prevention of thromboembolic conditions. However, coumarins are known to have a narrow therapeutic window and a considerable risk of bleeding complications caused by a broad variation of intra- and inter-individual drug requirement. Now, 3 years after its identification, VKORC1 has greatly improved our understanding of the vitamin K cycle and has led to the translation of basic research into clinical practise in at least three directions: (i) Mutations within VKORC1 have been shown to cause a coumarin-resistant phenotype and a single SNP (rs9923231) within the VKORC1 promoter region has been identified as the major pharmacodynamic determinant of coumarin dose. Together with the previously described CYP2C9 variants and other dose-influencing factors, such as age, gender and weight, individualized dosing algorithms have become available. (ii) Preliminary studies indicate that concomitant application of low-dose vitamin K (80-100 microg day(-1)) and warfarin significantly improves INR stability and time of INR within the therapeutic range. (iii) Co-expression studies of FIX and FX with VKORC1 have shown that VKOR activity is the rate-limiting step in the synthesis of biologically active vitamin K-dependent factors. Thus, co-expression of VKORC1 leads to a more efficient production of recombinant vitamin K-dependent coagulation factors such as FIX and FVII. This could improve production of recombinant factor concentrates in the future.

VKCFD2 - from clinical phenotype to molecular mechanism.

Vitamin K 2,3-epoxide reductase complex, subunit 1 (VKORC1) is an enzyme essential for the vitamin K cycle. VKORC1 catalyses the reduction of vitamin K 2,3-epoxide to the quinone form of vitamin K and further to vitamin K hydroquinone. The generated vitamin K hydroquinone serves as substrate for the enzyme γ-glutamyl-carboxylase which modifies all vitamin K-dependent proteins, allowing them to bind calcium ions necessary for physiological activity. Vitamin K-dependent proteins include the coagulation factors FII, FVII, FIX, FX, and proteins C, S und Z. Insufficient VKORC1 enzyme activity results in deficiency of the vitamin K-dependent clotting factors leading to haemorrhagic disorders. This phenotype is known as vitamin K clotting factor deficiency type 2 (VKCFD2). Worldwide, only four families of independent origin have been reported with this rare bleeding disorder. Affected family members carry the mutation VKORC1:p.Arg98Trp in homozygous form, the only mutation found so far to be associated with VKCFD2. Now, ten years after the identification of the VKORC1 gene, the molecular pathomechanism of VKCFD2 has been clarified. The Arg98Trp mutation disrupts an ER retention motif of VKORC1 leading to mislocalisation of the protein to outside the endoplasmatic reticulum. In this review, we summarize the clinical data, diagnosis, therapy and molecular pathomechanism of VKCFD2.

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.

BACKGROUND: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. RESULTS: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. CONCLUSIONS: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.

A rapid and sensitive protocol for competitive reverse transcriptase (cRT) PCR analysis of cellular genes.

The specific analysis of gene transcripts is of increasing importance for studies in molecular pathology. Competitive RT-PCR with mutagenized exogenous competitor templates has evolved as an attractive approach to quantify individual mRNA levels. The generation of exogenous competitor RNAs usually requires mutagenesis and cloning of the mutant fragment into plasmids followed by in vitro transcription. In contrast to primer directed mutagenesis and in vitro transcription, preparation of the mutant fragments is a time consuming procedure. Here we report on a modified semi-quantitative RT-PCR protocol to circumvent the laborious cloning of mutant exogenous competitors. Templates for the in vitro transcription are generated in a single PCR reaction with simultaneous addition of a promoter sequence 5'of the forward primer and deletion of 10-20 nucleotides at the opposite end just ahead of the reverse primer binding site. The product of this PCR step serves as template for in vitro transcription to yield exogenous competitor RNA of equal quality and amount as conventional cloning strategies. Total RNA amounts are corrected for by analyzing the expression of different housekeeping genes in the same manner. One of the primers used in the following competitive RT-PCR reaction is labeled with a fluorescent dye for the analysis of target and exogenous competitor product on an semiautomated sequencer. In the present study, this protocol was employed to analyze the expression of the PTCH, Fas-receptor, NF-1, beta2-microglobulin and GAPD genes in human brain tumors. It will, however, be widely applicable to studies on cellular transcripts in biological specimens.

Expression of 5alpha-reductase in the human temporal lobe of children and adults.

Androgens exert important biological effects on the brain, and 5alpha-reductase plays a crucial role in androgen metabolism. Therefore, we investigated the expression of the two isozymes of 5alpha-reductase in the human temporal lobe to determine the predominant isoform and to elucidate the existence of possible sex differences and differences between children and adults. We studied biopsy materials from the temporal lobe of 34 women, 32 men, and 12 children. Quantification of 5alpha-reductase 1 and 2 messenger ribonucleic acid (mRNA) was achieved by competitive RT-PCR. 5Alpha-reductase activity was determined in tissue homogenates using [1,2-3H]androstenedione as the substrate. Only 5alpha-reductase 1 mRNA was expressed in human temporal lobe tissue; 5alpha-reductase 2 mRNA was not expressed. 5Alpha-reductase 1 mRNA concentrations did not differ significantly in the cerebral cortex of women [25.9+/-7.9 arbitrary units (aU); mean +/-SEM] and men (20.4+/-2.8 aU) or in the cerebral cortex (23.3+/-4.4 aU) and the subcortical white matter of adults (32.6+/-5.6 aU), but they were significantly higher in the cerebral cortex of adults than in that of children (6.4+/-2.3 aU; P < 0.005). The apparent Km of 5alpha-reduction did not show significant differences between the two sexes. In conclusion, 5alpha-reductase 1 mRNA is expressed in the temporal lobe of children and adults, but 5alpha-reductase 2 mRNA is not. 5Alpha-reductase 1 mRNA concentrations did not differ significantly in the sexes, but they were significantly higher in specimens of adults than in those of children.

Characterization of the 5alpha-reductase-3alpha-hydroxysteroid dehydrogenase complex in the human brain.

Although androgen metabolism in the human brain was discovered almost 30 yr ago, conclusive studies on the enzymes involved are still lacking. We therefore investigated 5alpha-reductase and colocalized 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) activity in cerebral neocortex (CX) and subcortical white matter (SC) specimens neurosurgically removed from 44 patients suffering from epilepsy. We could demonstrate the presence of the 5alpha-reductase-3alpha-HSD complex in the biopsies of all patients under investigation. Inhibition experiments with specific inhibitors for 5alpha-reductase type 1 and type 2 revealed strong evidence for the exclusive activity of the type 1 isoform. We detected a significantly higher 5alpha-reductase activity in CX than in SC (P< 0.0001), but no sex-specific differences were observed. Furthermore, we found that, in contrast to liver, only 3alpha-HSD type 2 messenger RNA is expressed in the brain and that its expression is significantly higher in SC than in CX without sex-specific differences. The present study is the first to systematically characterize the 5alpha-reductase-3alpha-HSD complex in the human brain. The lack of sex-specific differences and also the colocalization of both enzymes at all life stages suggest a more general purpose of the complex, e.g. the synthesis of neuroactive steroids or the catabolism of neurotoxic steroids, rather than control of reproductive functions.

Reduced oxidative stress during acellular reperfusion of the rat liver after hypothermic oscillating perfusion.

BACKGROUND: ATP resynthesis during reperfusion after liver preservation has been shown to be well correlated with the function of transplanted grafts. Nevertheless, the advantages of a cellular energy charge loading during the preservation period are yet not fully understood. This study evaluates the effects of different nucleotide levels at the end of preservation on metabolic changes and oxidative stress during reperfusion. METHODS: Two experimental groups were chosen reflecting different energy charge states after preservation: static cold storage for 10 hr and hypothermic oxygenated oscillating perfusion for 10 hr. In both experimental groups, normothermic ex vivo acellular reperfusion over 40 min was performed. A third group consisted of nonpreserved livers similarly reperfused for 40 min. Superoxide formation was detected by the superoxide dismutase inhibitable reduction of ferricytochrome c added to the normothermic perfusate. RESULTS: Superoxide formation and lipid peroxidation malondialdehyde were significantly lower during reperfusion after the energy charge loading before reperfusion by the hypothermic oscillating perfusion technique. However, oxygen radical formation, liver cell injury (lactate dehydrogenase [LDH] release), and TNFalpha release were significantly higher in energy charge-depleted groups (nonpreserved and cold stored livers). CONCLUSIONS: Hypothermic oscillating oxygenated perfusion led to the elevated energy charge during preservation and led to reduced oxygen radical formation as well as less lipid peroxidation during reperfusion, in contrast to cold stored livers and nonpreserved livers. This suggests a correlation between the energy charge before reperfusion and oxygen radical formation as well as liver injury at reperfusion.

Expression of 17beta-hydroxysteroid dehydrogenase types 1, 2, 3 and 4 in the human temporal lobe.

Sex steroid hormones exert important biological effects on the brain. Moreover, an extensive sex steroid metabolism occurs in the brain. In sex steroid metabolism 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play essential roles in catalyzing the final steps in androgen and estrogen biosynthesis. Recently four types of human 17beta-HSDs and a pseudogene of the type 1 isoform were identified. To date, 17beta-HSD has not been extensively studied in the human brain. Therefore, we investigated the mRNA expression of the four isozymes of 17beta-HSD as well as the pseudogene of the type 1 isoform in the human temporal lobe to determine the predominant isoforms and, moreover, to elucidate the existence of possible sex and age differences. We studied biopsy materials from the temporal lobe of 34 women, 32 men and 10 children. Quantification of different mRNAs was achieved by competitive reverse transcription-PCR. 17beta-HSD 1, 17beta-HSD 3 and 17beta-HSD 4 were expressed in the human temporal lobe of children and adults, whereas 17beta-HSD 2 and the pseudogene of 17beta-HSD 1 were not expressed. In adults, 17beta-HSD 3 and 17beta-HSD 4 mRNA concentrations were significantly higher in the subcortical white matter (17beta-HSD 3: 14 591+/-3457 arbitrary units (aU), mean+/-s.e.m.; 17beta-HSD 4: 1201+/-212 aU) than in the cortex (17beta-HSD 3: 5428+/-1057 aU, P<0. 0002; 17beta-HSD 4: 675+/-74 aU, P<0.004). 17beta-HSD 1 concentrations did not differ significantly between the white matter (3860+/-1628 aU) and the cortex (2525+/-398 aU) of adults. In conclusion, the present study demonstrates the expression of 17beta-HSD 1, 3 and 4 mRNAs in the human temporal lobe. Together with CYP19AROM and 5alpha-reductase, known to be expressed in the human brain, the expression of 17beta-HSD 1, 3 and 4 mRNAs indicates the major importance of local steroid biosynthesis in the brain.

Expression of the 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the human brain.

An enzyme-mediated metabolism of androgens and estrogens including 17beta-HSD activity in the brain of vertebrates was discovered approximately 30 years ago. Mainly 5alpha-reductase and aromatase have been studied in detail. Recently we could demonstrate reductive and oxidative 17beta-HSD activity as well as considerable mRNA expression of the 17beta-HSD types 3 and 4 in the human brain. In the present study, we report on 17beta-HSD type 5 mRNA expression in brain tissue of women and men. Data analysis did not reveal sex specific differences, but we determined a significantly higher mRNA concentration in the subcortical white matter (SC) than in the cerebral cortex (CX). Investigation of reductive 17beta-HSD in vitro activity with 2 microM androstenedione as the substrate revealed no sex specific differences. Testosterone formation was significantly higher in SC than in CX. Moreover, enzyme activity was significantly higher in brain tissue of adults compared to that of children.

Mineralocorticoid receptor splice variants in different human tissues.

The mineralocorticoid receptor (MR), a member of the steroid receptor family, acts as a transcription factor and mediates both aldosterone and cortisol effects. Aldosterone specificity in some tissues results from the inactivation of competing cortisol into cortisone by 11beta-hydroxysteroid dehydrogenase. In other tissues MR and the glucocorticoid receptor show overlapping physiological effects or may act together by forming a heterodimer. An additional MR splice variant (MR+4) has been found in different mRNA samples from rat tissues and human white blood cells, thereby implying additional modes of MR-regulated effects. We therefore looked for the presence of these two MR-mRNA isoforms in human classical aldosterone target tissues and various other tissues. MR-mRNA was found in all samples investigated, thereby showing the expression of MR to be more abundant than has been observed thus far. In addition, the MR+4-mRNA variant was also found in all the tissues examined.

Sex- and age-specific differences in human brain CYP11A1 mRNA expression.

While the presence of CYP11A1 (P450SCC, cholesterol side-chain cleavage enzyme) has been well established in the brain of rodents, limited information is available on CYP11A1 expression in human brain. In both species, little is known regarding postnatal changes or sex specific differences in cerebral CYP11A1 expression. In the present study, we used a sensitive competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay to quantify the amount of CYP11A1 mRNA in a large number of human brain tissue specimens obtained at neurosurgery. CYP11A1 mRNA is expressed approximately 200 times lower in the temporal lobe, frontal lobe and hippocampus than in adrenal tissue, known for high CYP11A1 mRNA expression. During childhood CYP11A1 mRNA concentrations in the temporal lobe increase markedly and reach adult levels at puberty. CYP11A1 mRNA is significantly higher in the temporal and frontal lobe cortex of women than in that of men. Our data demonstrate for the first time an age and sex dependent expression of CYP11A1 mRNA in the human brain.

Differential mRNA expression of the two mineralocorticoid receptor splice variants within the human brain: structure analysis of their different DNA binding domains.

In human brain tissue, cortisol action, at basal concentrations, is mediated by the mineralocorticoid receptor (MR). An in-frame insertion of 12 bp in the MR-DNA-binding domain due to alternative splice site usage between exons 3 and 4 results in an MR mRNA splice variant (MR+4) encoding a receptor protein with four additional amino acids compared to the wild-type MR protein. To elucidate the questions of sex, age, and/or tissue dependent differences of the relative amount of the two mRNA subtypes, we examined 131 fresh human brain tissue samples from temporal and frontal lobe or hippocampus. One hundred and twenty samples were obtained from patients with epilepsy and 11 samples from patients with brain tumours. A small but significant difference of the MR+4 mRNA splice variant proportions in cortex (9.5 +/- 0.8%) and subcortical white matter (6.6 +/- 0.7%) of the temporal lobe could be detected, indicating differential MR splice variant expression within these brain areas. Moreover, the splice variant ratios in samples of the temporal lobe cortex collected from patients with epilepsy differed from samples of patients with brain tumours. These data point to an altered expression of the MR splice variants in epilepsy, and strengthen the supposition of a tissue specific alternative splicing of the MR mRNA. The frequent occurrence of the MR+4 transcript raises the question of its functional significance. For this reason, an MR+4 DNA-binding-domain structure model was generated by computer-based homology modelling based on the known glucocorticoid receptor structure. The data obtained revealed no distorting effect of the inserted four amino acids on the adjacent secondary structures, thereby suggesting that both zinc fingers retain their function. The resulting structure of the MR+4 model leads to the supposition that the receptor retains its function. Moreover, databank analysis with respect to this kind of steroid receptor variation and our own sequence data of the closely related progesterone receptor sustained the hypothesis that only corticosteroid receptors were affected by this alternative splicing event.

Expression of CYP19 (aromatase) mRNA in different areas of the human brain.

The conversion of androgens to estrogens by CYP19 (cytochrome P450AROM, aromatase) is an important step in the mechanism of androgen action in the brain. CYP19 expression has been demonstrated in the brain of various animal species and in the human temporal lobe. Studies on postnatal CYP19 expression in various other areas of the human brain are rare and carried out in a limited number of post mortem obtained tissue. Therefore, we investigated CYP19 mRNA expression in fresh human frontal and hippocampal tissues and compared them to the expression in temporal neocortex tissues. We studied biopsy materials removed at neurosurgery from 45 women and 54 men with epilepsy. Quantification of CYP19 mRNA was achieved by nested competitive reverse transcription-PCR. CYP19 mRNA concentrations were significantly higher in temporal (2.29+/-0.40 arbitrary units, AU, mean +/- SEM; n = 57) than in frontal neocortex specimens (0.92+/-0.17 AU; n = 18; P<0.04). In hippocampal tissue specimens CYP19 expression (1.41+/-0.18 AU; n = 24) was lower than in temporal neocortex specimens, but the difference did not reach statistical significance. Sex differences were not observed in any of the brain regions under investigation. In conclusion, CYP19 mRNA is expressed in the human temporal and frontal neocortex as well as in the hippocampus. Regardless of sex, CYP19 expression was significantly higher in the temporal than in the frontal neocortex.

Expression of cytochrome P450scc mRNA in the hippocampus of patients with temporal lobe epilepsy.

The hippocampus is one of the target areas of neurosteroidal action. Expression of cytochrome P450scc (P450scc, CYP11A1), one of the key enzymes in steroid metabolism, results in de novo synthesis of the neurosteroid pregnenolone. We used a competitive RT-PCR assay to quantify the amount of P450scc mRNA in hippocampal tissue specimens obtained at neurosurgery from patients with temporal lobe epilepsy (TLE). P450scc mRNA is expressed approximately 200 times lower in the hippocampus than in adrenal tissue known for high P450scc expression. P450scc mRNA concentrations were significantly higher in the hippocampus of women (1.72 +/- 0.36 aU, arbitrary units; mean +/- s.e.m.) than of men (0.92 +/- 0.15 aU, p < 0.004). Our data show for the first time the sex-dependent expression of P450scc mRNA in the hippocampus of patients with TLE.

Androgen receptor mRNA expression in the human hippocampus.

The androgen receptor (AR) plays a central role in mediating androgen action. Since the hippocampus is a target of steroid modulation, we studied the expression of AR mRNAs in hippocampal tissue specimens from patients undergoing epilepsy surgery (n=42). AR mRNA expression was in the same order of magnitude than in prostate tissue, known for its high expression of AR. AR mRNA concentrations showed no significant difference in AR mRNA expression between men (49.3+/-8.0 arbitrary units (aU); mean+/-SEM) and women (54.3+/-11.2 aU) and no sex-specific hippocampal lateralization pattern was observed. No relationship could be detected between duration of epilepsy, individual seizure frequency, age of the patients and the expression levels of AR. The high expression of AR in the hippocampus suggests that this human brain area is an important target for androgen action.

Expression of mineralocorticoid and glucocorticoid receptor mRNA in the human hippocampus.

The genomic effects of corticosteroids in the brain are mediated through two receptors with a high affinity for cortisol: the glucocorticoid and mineralocorticoid receptor (GR/MR). We used competitive reverse transcription-polymerase chain reaction to quantify the amount of MR and GR mRNA in hippocampal tissue obtained from patients with temporal lobe epilepsy. MR and GR mRNA were expressed at approximately the same levels as in tissues known for high glucocorticoid/mineralocorticoid sensitivity, i.e. liver or kidney. MR mRNA concentrations were significantly higher in the hippocampus of women (0.24+/-0.04 aU, arbitrary units; mean+/-SEM) than in men (0.14+/-0.01 aU, P<0.006) or children (0.09+/-0.02, P<0. 007). No such differences were observed for GR mRNA expression.

Messenger RNA of steroid 21-hydroxylase (CYP21) is expressed in the human hippocampus.

21-hydroxylase converts progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to 11-deoxycortisol, the substrates which are required for the production of the main adrenal steroids, corticosterone, aldosterone, and cortisol. As 21-hydroxylase activity has been detected in rodent and fetal human brain, we studied whether and to what extent 21-hydroxylase mRNA is expressed in hippocampal tissue specimens from patients undergoing epilepsy surgery (n=42). 21-hydroxylase mRNA was detected in the hippocampus with an expression 10 000 times lower than in adrenal gland tissue. There was no significant difference in expression levels between women (9.5+/-2.7 arbitrary units (aU); mean+/-SEM) and men (8.0+/-2.2 aU); however, mRNA concentrations in the hippocampus of children (n=4, 1.8+/-0.5 aU) were considerably lower than in adults (n=38, 8.6+/-1.7 aU). The expression of 21-hydroxylase mRNA in the hippocampus suggests that this human brain area has the enzymatic capability to convert progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to 11-deoxycortisol.

Neuroactive steroids and seizure susceptibility.

There is increasing clinical and experimental evidence that hormones, in particular sex steroid hormones, influence neuronal excitability and other brain functions. The term 'neuroactive steroids' has been coined for steroids that interact with neurotransmitter receptors. One of the best characterized actions of neuroactive steroids is the allosteric modulation of GABA(A)-receptor function via binding to a putative steroid-binding site. Since neuroactive steroids may interact with a variety of other membrane receptors, excitatory as well as inhibitory, they may have an impact on the excitability of specific brain regions. Neuronal excitability is enhanced by estrogen, whereas progesterone and its metabolites exert anticonvulsant effects. Testosterone and corticosteroids have less consistent effects on seizure susceptibility. Apart from these particular properties, neuroactive steroids may regulate gene expression via progesterone receptors. Based on their molecular properties, these compounds appear to have a promising therapeutical profile for the treatment of different neuropsychiatric diseases including epilepsy. This review focuses on the effects of neuroactive steroids on neuronal excitability and their putative impact on the physiology of epileptic disorders.

Expression of mRNAs encoding for 17beta-hydroxisteroid dehydrogenase isozymes 1, 2, 3 and 4 in epileptic human hippocampus.

Sex steroid hormones exert important influences on neuroendocrine and behavioural brain function. As neuroactive steroids they are able to modify neuronal excitability. Unbalanced synthesis may thus be implicated in pathophysiological conditions, such as epilepsy, migraine, depression and anxiety. In sex steroid metabolism, 17beta-hydroxisteroid dehydrogenases (17beta-HSDs) play a crucial role in catalyzing the final steps of androgen and estrogen biosynthesis. The hippocampus appears to be a major target area of neurosteroidal action. The expression of 17beta-HSD isozymes has not yet been studied in human hippocampus. Therefore, we investigated the expression of 17beta-HSD 1, 2, 3 and 4 mRNAs in hippocampal tissue specimens obtained at neurosurgery from 42 patients with pharmacoresistant temporal lobe epilepsy. A competitive RT-PCR assay was used to quantify the mRNA transcript level. 17beta-HSD 1 mRNA concentrations were 10000 fold lower in the hippocampus compared to placental tissue, whereas 17beta-HSD 3 mRNA concentrations were 50 fold lower than in testis and 17beta-HSD 4 concentrations were in the same order of magnitude as in liver. 17beta-HSD 2 mRNA was not expressed. 17beta-HSD 1, 3 and 4 mRNA concentrations in the hippocampus showed no significant differences between men and women and there were no significant differences in expression levels of these enzymes between patients with Ammon's horn sclerosis (AHS) and those with histopathologically normal hippocampus associated with extrahippocampal lesions. No significant correlation could be detected between duration of epilepsy, individual seizure frequency and expression levels of 17beta-HSDs. In conclusion, the present study is the first to demonstrate mRNA expression of 17beta-HSD 1, 3 and 4 in the epileptic human hippocampus. Together with data on 5alpha-reductase 1, 3alpha-hydroxisteroid oxidoreductase 2 and cytochrome P450scc, previously shown to be expressed in the human hippocampus also, our data provide further evidence for the existence of sex steroid formation and metabolism in this specific brain area.