Specific and irreversible cyclopeptide inhibitors of dipeptidyl peptidase IV activity of the T-cell activation antigen CD26

The dipeptidyl peptidase IV (DPP IV) activity of CD26 is characterized by its post-proline-cleaving capacity that plays an important but not yet understood role in biological processes. Here we describe a new family of specific and irreversible inhibitors of this enzyme. Taking into account the substrate specificity of DPP IV for P2-P1><-P1' cleavage, we have designed and synthesized cyclopeptides c[(alphaH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn] 2TFA- (Aba = 3-aminobenzoic acid, R = alkyl) possessing a proline at the P1 position and a lysine in the P2 position, which allows the closing of the cycle on its side chain. These molecules show a free N-terminus, necessary for binding to the CD26 catalytic site, and a latent quinoniminium methide electrophile, responsible for inactivation. Treatment of c[alphaZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn], obtained by peptide synthesis in solution, with R2S/TFA simutaneously cleaved the Z protecting group and the phenyl ether function and led to a series of cyclopeptide sulfonium salts. These cyclopeptides inhibited rapidly and irreversibly the DPP IV activity of CD26, with IC50 values in the nanomolar range. Further studies were carried out to investigate the effect of the modification of the ring size (n = 2 or 4) and the nature of the sulfur substituents (R = Me, Bu, Oct). Cycle enlargement improved the inhibitory activity of the methylsulfonio cyclopeptide, whereas the increase of the alkyl chain length on the sulfur atom had no apparent effect. Other aminopeptidases were not inhibited, and a much weaker activity was observed on a novel isoform of DPP IV referred to as DPP IV-beta. Thus, this new family of irreversible inhibitors of DPP IV is highly specific to the peptidase activity of CD26.     

Lower serum dipeptidyl peptidase IV activity in treatment resistant major depression: relationships with immune-inflammatory markers

Previous research in this laboratory has shown that major depression is accompanied by decreased serum activity of dipeptidyl peptidase IV (DPP IV), a serine protease that cleaves N terminal dipeptides from peptides with penultimate proline or alanine. DPP IV is involved in the metabolism of peptides, T cell activation and proliferation, including the production of cytokines, such as interleukin-1 (IL-1) and IL-2. The aim of this study was to examine (i) serum DPP IV activity in major and treatment resistant depression (TRD) in relation to other established immune and inflammatory markers of that illness, and (ii) the effects of antidepressive treatment on DPP IV activity. Serum DPP IV activity was significantly lower in major depression and TRD than in normal controls. In normal and major depressed subjects, there were significant and positive relationships between serum DPP IV activity and total serum protein, serum albumin, zinc, iron and transferrin. In the group of depressed subjects, there were significant and positive relationships between serum DPP IV activity and number of CD4+T cells and CD4+/CD8+ T cell ratio. There were no significant effects of subchronic treatment with antidepressants on serum DPP IV activity. The findings suggest that: (i) lower serum DPP activity may occur in chronic depression, TRD as well as in the acute phase of major depression; (ii) lower serum DPP IV accompanies the 'chronic' acute phase response in depression; and (iii) serum DPP IV activity is tightly coupled to increased number of CD4+ T cells in depressed subjects, but not in normal controls. Our results do not exclude the possible effects of longer-term treatment with antidepressants on serum DPP-IV activity.     

Truncation of macrophage-derived chemokine by CD26/ dipeptidyl-peptidase IV beyond its predicted cleavage site affects chemotactic activity and CC chemokine receptor 4 interaction

The serine protease CD26/dipeptidyl-peptidase IV (CD26/DPP IV) and chemokines are known key players in immunological processes. Surprisingly, CD26/DPP IV not only removed the expected Gly1-Pro2 dipeptide from the NH2 terminus of macrophage-derived chemokine (MDC) but subsequently also the Tyr3-Gly4 dipeptide, generating MDC(5-69). This second cleavage after a Gly residue demonstrated that the substrate specificity of this protease is less restricted than anticipated. The unusual processing of MDC by CD26/DPP IV was confirmed on the synthetic peptides GPYGANMED (MDC(1-9)) and YGANMED (MDC(3-9)). Compared with intact MDC(1-69), CD26/DPP IV-processed MDC(5-69) had reduced chemotactic activity on lymphocytes and monocyte-derived dendritic cells, showed impaired mobilization of intracellular Ca2+ through CC chemokine receptor 4 (CCR4), and was unable to desensitize for MDC-induced Ca2+-responses in CCR4 transfectants. However, MDC(5-69) remained equally chemotactic as intact MDC(1-69) on monocytes. In contrast to the reduced binding to lymphocytes and CCR4 transfectants, MDC(5-69) retained its binding properties to monocytes and its anti-HIV-1 activity. Thus, NH2-terminal truncation of MDC by CD26/DPP IV has profound biological consequences and may be an important regulatory mechanism during the migration of Th2 lymphocytes and dendritic cells to germinal centers and to sites of inflammation.     

Heart Partners: a strategy for promoting effective diffusion of school health promotion programs

To investigate the mechanism whereby serum dipeptidyl peptidase (DPP) IV activity in oral cancer patients is decreased, we examined the expression of cell surface DPP IV, also known as CD26, in cultured peripheral blood T lymphocytes of these patients and the amounts of DPP IV released into culture medium; values were compared with those found in healthy subjects. When peripheral blood T lymphocytes were cultured in the presence of phytohemagglutinin, concanavalin A and/or interleukin-2, the proliferative response and expression of CD26 (DPP IV) in their plasma membranes were greatly diminished in oral cancer patients as compared with those in healthy subjects. In addition, DPP IV activity in lymphocyte culture medium was reduced more in oral cancer patients than in healthy subjects, indicating decreased shedding of DPP IV from activated T lymphocytes in the patients. Based on these findings, it is suggested that suppression of DPP IV expression in peripheral blood T lymphocytes is one of the important factors involved in the mechanism of decrease of serum DPP IV activity in oral cancer patients.     

Dipeptidyl-peptidase IV-beta, a novel form of cell-surface-expressed protein with dipeptidyl-peptidase IV activity

The T-cell activation antigen CD26, is a type II membrane glycoprotein with intrinsic dipeptidyl-peptidase IV (DPP IV) activity, characterized by its capacity to cleave off N-terminal dipeptides containing proline as the penultimate residue. Independent of its catalytic activity, CD26 has also been characterized as adenosine deaminase binding protein. By using CD26 negative human C8166 cells, here we describe the existence of another cell-surface protein which manifests CD26-like DPP IV activity. For convenience, this protein will be referred to as DPP IV-beta. Consistent with the cell-surface expression of DPP IV-beta, intact C8166 cells manifested a high level of DPP IV, whereas, they manifested poor activity against substrates of DPP II known to have an intracellular localization. A partially purified preparation of CD26 from human MOLT4 cells, and the DPP IV-beta expressed on intact cells were found to possess similar catalytic activity and pH optimum. In addition, cell-surface CD26 and DPP IV-beta on intact MOLT4 and C8166 cells, respectively, resisted digestion by proteolytic enzymes such as trypsin and proteinase K. However, adenosine deaminase activity was not detectable on the surface of C8166 cells in contrast to CD26 positive MOLT4 cells. In accord with this, 125I-labeled adenosine deaminase which binds CD26 was found not to bind DPP IV-beta. Gel-filtration experiments using 0.5% Triton X-100 extracts from C8166 and MOLT4 cells, revealed that the apparent molecular mass of DPP IV-beta is 82 kDa, whereas that of CD26 is 110 kDa as expected. Taken together, our results suggest that DPP IV-beta is a CD26-like protein which could be characterized by distinct properties.     

The hypnic ("alarm clock") headache syndrome

Dipeptidyl peptidase IV (DPP IV, CD 26) is an integral membrane serine protease exhibiting a well characterized exopeptidase activity. The present study shows that DPP IV also possesses a novel gelatinase activity and therefore endopeptidase activity, which was directly demonstrated by gelatin zymography. Protease inhibitor profile analysis showed that the endo- and exopeptidase activities of DPP IV share a common active site. Substrate specificity was detected for denatured collagen types I, II, III and V suggesting that DPP IV might contribute to collagen trimming and metabolism. On the basis of these data we propose that DPP IV and the recently sequenced gelatinolytic seprase (FAPalpha) represent a new subfamily of gelatinolytic integral membrane serine proteases.     

Dipeptidyl peptidase IV from human serum: purification, characterization, and N-terminal amino acid sequence

Dipeptidyl peptidase IV (DPP IV) in normal human serum was purified 14,400-fold with a 25% yield to homogeneity. The molecular weight of the purified enzyme was approximately 110,000 on SDS-PAGE, almost the same as that of human kidney membrane-bound DPP IV. No difference was found between the two enzymes enzymologically and immunologically, either in substrate specificity, susceptibility to inhibitors, or cross-reactivity with an anti-rat kidney DPP IV antibody, or in their ability to bind adenosine deaminase. However, the N-terminal amino acid sequence of serum DPP IV lacked the transmembrane domain of the membrane-bound enzyme and started at the 39th position, serine, from the N-terminus predicted from the cDNA nucleotide sequence. These results suggest that membrane-bound DPP IV loses its transmembrane domain upon release into the serum, and that its structure on the plasma membrane is not required for its binding to adenosine deaminase.     

Peptidases in human bronchoalveolar lining fluid, macrophages, and epithelial cells: dipeptidyl (amino)peptidase IV, aminopeptidase N, and dipeptidyl (carboxy)peptidase (angiotensin-converting enzyme)

The modulation of proteolytic activity is an important factor in regulating the metabolism and function of peptide hormones. In this study, the activities of dipeptidyl (carboxy)peptidase (angiotensin-converting enzyme [ACE]), aminopeptidase N (APN), and dipeptidyl (amino)peptidase IV (DPP IV) were measured in the blood, the human bronchial epithelial and alveolar cells, bronchoalveolar macrophages, and the soluble phase of bronchoalveolar lavage (BAL) samples obtained from normal human volunteers and patients with pulmonary pathologic conditions. BAL fluid expressed ACE activity and very low levels of APN and DPP IV activities in the volunteer population, but higher levels could be measured in samples from patients. In patients, increased APN corresponded to a high granulocyte count, while DPP IV and ACE were associated with a high percentage of lymphocytes. Neither AIDS nor smoking induced an increased level of these enzymes. Immunohistochemical staining of bronchoalveolar smears with anti-human ACE monoclonal antibody showed that only macrophages expressed this enzyme. Enzyme histochemistry for DPP IV and APN showed that all leukocytes expressed these activities. APN, DPP IV, and ACE activities were also found in cell extracts of bronchoalveolar macrophages. In extracts of bronchial epithelial and alveolar cells, only APN and DPP IV activities were detected. Kinetic properties of the soluble enzymes in lavage supernatants were comparable to those of serum enzymes. These results demonstrate that soluble forms of cellular enzymes found in BAL fluid are regulated independently of blood and that different cell types may release these enzymes.     

A potent dipeptide inhibitor of dipeptidyl peptidase IV

A series of novel potent inhibitors of dipeptidyl peptidase IV (DPP-IV) has been developed. A brief structure-activity relationship of the inhibitors was investigated. The dipeptide TSL-225, tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, was identified with the critical structure for the inhibitory activity.     

Dipeptidyl peptidase IV from porcine seminal plasma: purification, characterization, and N-terminal amino acid sequence

Dipeptidyl peptidase IV (DPP IV) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approximately 290,000 on PAGE in the absence of sodium dodecyl sulfate (SDS) and 310,000 on Sephacryl S-300 HR column chromatography, and to be 115,000 and 105,000 on SDS-PAGE in the absence and presence of beta-mercaptoethanol. The enzyme is suggested to be composed of three identical subunits. The enzyme rapidly hydrolyzed the substrate Gly-Pro-MCA, and weakly the substrate Lys-Ala-MCA. It was strongly inhibited by diisopropylphosphofluoridate (DFP), and moderately by both phenylmethyl-sulfonyl fluoride (PMSF) and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). It was also strongly inhibited by zinc ion. The amino acid sequence of the first 18 residues of the enzyme was Asn-Lys-Gly-Thr-Asp-Asp-Ala-Ala-Ala-Asp-Ser-Arg-Arg- Thr-Tyr-Thr-Leu-Thr-. This sequence was highly homologous to the sequences in the rear of the transmembrane site of human and rat liver DPP IVs and mouse thymus DPP IV. The native DPP IV is suggested to be released into the seminal plasma after the cleavage of the hydrophobic N-terminal domain by chymotrypsin-like or pepsin-like enzymes. Other properties of DPP IV including kinetic parameters, pH stability and heat stability were characterized.     

Modulation of endomorphin-2-induced analgesia by dipeptidyl peptidase IV

The tetrapeptide, endomorphin-2 (Tyr-Pro-Phe-PheNH2) possesses high affinity for mu opioid receptors, and produces potent analgesia in mice. Its structure appears to satisfy the substrate requirements of the proteinase, dipeptidyl peptidase IV which removes dipeptides from the amino terminus of peptides containing proline as the penultimate amino acid. A potent, stable and specific inhibitor of this enzyme, Ala-Pyrrolidonyl-2-nitrile, has been described which should potentiate endomorphin-2-induced analgesia. Further, since dipeptidyl peptidase IV has an absolute requirement for l-Pro, a more metabolically-stable d-Pro2-endomorphin-2 analog should produce longer analgesic actions at lower doses. The present study found that endomorphin-2 was degraded approximately twice as fast than the chromogenic substrate, Ala-Pro-2naphthylamide, by dipeptidyl peptidase IV, whereas d-Pro2-endomorphin-2 was totally resistant to this enzyme's action. d-Pro2-endomorphin-2 (ED50=0.05 microg) was more potent than endomorphin-2 (ED50=30 microg) in significantly increasing tail-flick latencies with longer durations of action. Both the peptide and analogue were equipotent (ED50=0.5 microg) in significantly increasing jump thresholds. Ala-Pyrrolidonyl-2-nitrile (10-75 nmol) elicited a dose-dependent analgesia, and potentiated the analgesic actions of endomorphin-2, particularly on the tail-flick test. Whereas systemic naltrexone (2.5, 10 mg/kg) dose-dependently eliminated each of the three forms of analgesia on the jump test as well as the peak (15 min) effect on the tail-flick test, analgesia elicited by either endomorphin-2, d-Pro2-endomorphin-2 or Ala-Pyrrolidonyl-2-nitrile returned after 30-60 min in naltrexone-treated rats on the tail-flick test. These data strongly suggest that dipeptidyl peptidase IV plays a role in the inactivation of endomorphin-2 in vivo, and thereby modulates its central analgesic actions.     

Biochemical properties of human oral polymorphonuclear leukocytes

There is now some evidence that psychiatric disorders, such as major depression, schizophrenia and post-traumatic stress disorder are associated with significant alterations in the serum activity of peptidases, such as prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPP IV). The aims of the present study were to examine the effects of psychological stress on serum PEP and DPP IV activity in humans. Thirty-eight university students had repeated measurements of serum PEP and DPP IV activity a few weeks before and after (baseline conditions) as well as the day before a difficult academic examination (stress condition). Subjects were divided into anxiety responders and nonresponders to stress according to their stress-induced increase in the Spielberger State Anxiety Inventory. Serum PEP activity was somewhat lowered by stress in female, but not male, students. Serum PEP activity was significantly higher in the two baseline conditions and during the stress condition in anxiety responders than in anxiety nonresponders. There were no significant effects of stress on serum DPP IV activity and no significant differences between anxiety responders and nonresponders. Serum PEP and DPP IV activity were significantly higher in men than in women. The results suggest that increased baseline serum PEP activity is related to stress-induced anxiety.     

Prostate specific origin of dipeptidylpeptidase IV (CD-26) in human seminal plasma

PURPOSE: A number of peptidases which can metabolize certain bioactive peptides and growth factors have been identified in seminal plasma. Our goal in this study was to determine molecular properties and the tissue source(s) for one of these peptidases, dipeptidylpeptidase IV (DPP IV), in human seminal plasma. MATERIALS AND METHODS: We measured the activities of DPP IV with the dipeptide glycylprolyl-p-nitroanalide and its molecular forms using immunoblotting of seminal plasmas of men who were vasectomized or with different sperm concentrations, and in prostatic and seminal vesicle secretions of men undergoing prostatic surgery. RESULTS: DPP IV in seminal plasma of vasectomized men was a membrane associated dimer comprised of subunits of approximately 110 kDa. Its activity did not differ in seminal plasmas of vasectomized, azoospermic, oligozoospermic and normozoospermic men indicating no correlation with the concentration of sperm originally present in the semen. The DPP IV antigen (CD -26) and enzymic activity were present in prostatic secretion, but absent from that of the seminal vesicles. These data indicate that the prostate gland is the primary source of DPP IV activity in seminal plasma. There was little variation in its activities in repeat seminal plasma samples from the same individual, and there was no change in its activity with age to 50 years. CONCLUSIONS: DPP IV in seminal plasma was derived from the prostate gland and it may be useful as a bioindicator of prostate function and/or disease with age in men.     

The Mycobacterium tuberculosis phagosome interacts with early endosomes and is accessible to exogenously administered transferrin

CD26, a T cell activation Ag, also known as dipeptidyl peptidase IV, is directly associated with adenosine deaminase (ADA) on the surface of T cells and T cell lines. In the present study, we examined both the binding of ADA and CD26 and the functional consequences of this interaction. We found that ADA was associated with CD26 on T cell lines lacking either ADA or dipeptidyl peptidase IV enzymatic activity, indicating that the association between dipeptidyl peptidase IV and ADA did not require enzymatic activity. Moreover, using immunoelectron microscopy, we demonstrated that CD26 and ADA co-localized on the cell surface, but not inside cells, suggesting that CD26 did not transport ADA to the surface. In keeping with this observation, we showed that human CD26-transfected murine pre-B cell lines lacking human ADA acquired ADA from an extracellular source. More importantly, adenosine in the absence of cell surface ADA inhibited T cell proliferation and IL-2 production induced by various stimuli. On the other hand, cells expressing ADA and CD26 on the surface were much more resistant to the inhibitory effect of adenosine. These data suggest that ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to cell surface CD26, and this complex is capable of reducing the local concentration of adenosine.     

Lymphocyte ectoenzymes in childhood idiopathic nephrotic syndrome

Lymphocyte ectoenzymes with immunomodulatory function were investigated in 11 children with minimal change disease (MCD), 9 with primary focal segmental glomerulosclerosis (FSGS), and 17 age- and sex-matched healthy children. Basal, concanavalin A (Con A)-, and pokeweed mitogen (PWM)-stimulated lymphocyte ecto-5'-nucleotidase (5'-Nu), dipeptidyl peptidase IV (DPP IV), and alkaline phosphodiesterase I (APD) activities were determined. In MCD relapse ecto-APD activity of unstimulated lymphocytes was higher than controls. Ecto-APD of Con A-stimulated lymphocytes was below controls (23.0, range 7.2-48.7 nmol/min per 10(6) lymphocytes) in all active MCD (18.7, range 7.6-32.6), during corticosteroid treatment (14.6, range 4.5-54), and in remission (13.1, range 6.1-19.6), but was significant only in remission. Con A-stimulated DPP IV was significantly lower from controls (53.8, range 19.3-85.7 nmol/min per 10(6) lymphocytes) in all active MCD (38.1, range 10.8-82.1), during treatment (37.5, range 20.2-58.7), and in remission (39.4, range 24.3-69.6). In FSGS, unstimulated lymphocyte ecto-APD activity was greater than controls. However, Con A-stimulated lymphocyte ecto-APD and DPP IV activities were not significantly different from controls. Con A stimulation of lymphocyte ecto-APD and DPP IV activity was significantly reduced in MCD relapse and in remission, but not in FSGS. Basal, Con A-, and PWM-stimulated ecto-5'-Nu in MCD and FSGS were not different from controls. These results suggest a role for abnormal T cell function in MCD but not in FSGS. The difference in mitogen-stimulated expression of these ectoenzymes suggests a different pathogenesis of childhood MCD and primary FSGS.     

Pathway for uptake and degradation of X-prolyl tripeptides in Streptococcus mutans VA-29R and Streptococcus sanguis ATCC 10556

The growth of Streptococcus mutans and Streptococcus sanguis in the oral environment requires that these micro-organisms be able to degrade salivary proteins and to assimilate the resulting peptides as an amino nitrogen source. Our research is aimed at the definition of the proteolytic enzyme systems in these oral streptococci which allow them to utilize such substrates. In the present work, the nature of the hydrolytic activity expressed by S. mutans VA-29R and S. sanguis ATCC 10556 against X-Pro4-nitroanilide and X-Pro-Y tripeptide substrates was investigated. This activity was predominantly associated with a cytoplasmic dipeptidyl peptidase which preferentially catalyzes the release of an N-terminal dipeptide from substrates in which proline is the penultimate residue. These streptococci also possess a second cytoplasmic peptidase, pepD, which catalyzes the hydrolysis of X-Pro dipeptides. We found that Gly-Pro-Ala or Ala-Pro-Gly were transported into the bacterial cells only when an energy source such as glucose was present. Peptide uptake was time-dependent, and selective exodus of peptide-derived amino acids from the bacterial cells occurred during peptide uptake. Results from these studies provide evidence that S. mutans VA-29R and S. sanguis ATCC 10556 possess a pathway for the complete degradation of X-Pro tripeptides. Transport of the peptides into cells prior to hydrolysis provides an efficient way by which all amino acids of a peptide may be obtained at an energy expense equivalent to that associated with the transport of just one amino acid. In light of the abundance of proline in salivary polypeptides, this degradative pathway could be an important component in the proteolytic pathway for salivary polypeptide utilization in these oral streptococci.     

Novel small renin inhibitors containing 4,5- or 3,5-dihydroxy-2-substituted-6-phenylhexanamide replacements at the P2-P3 sites

Renin inhibitors containing a 4,5- or a 3,5-dihydroxy-2-substituted-6-phenylhexanamide fragment at the P2-P3 sites have been prepared and evaluated. The four possible diastereomeric diols of the two series of inhibitors were synthesized to determine the optimal configuration of the carbinol centers for these replacements. The most potent inhibitors of each series, la and 2c have a molecular weight of only 503 and IC50 values of 23 and 20 nM in a human plasma renin assay at pH 6.0. Their very low aqueous solubility limited their further evaluation. The efficacy of these P2-P3 replacements is a result of their ability to maintain the important hydrogen-bonds with the enzyme. Due to conformational differences with the dipeptide, adjustment at the P2 side chain was required. These 4,5- and 3,5-dihydroxyhexanamide segments could be seen as novel N-terminal dipeptide replacements.     

Seven-day treatment for Helicobacter pylori infection: ranitidine bismuth citrate plus clarithromycin and tetracycline hydrochloride

1. We have examined a series of novel phosphinic peptides as putative potent and selective inhibitors of endopeptidase 3.4.24.16. 2. The most selective inhibitor, Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 displayed a Ki value of 12 nM towards endopeptidase 3.4.24.16 and was 5540 fold less potent on its related peptidase endopeptidase 3.4.24.15. Furthermore, this inhibitor was 12.5 less potent on angiotensin-converting enzyme and was unable to block endopeptidase 3.4.24.11, aminopeptidases B and M, dipeptidylaminopeptidase IV and proline endopeptidase. 3. The effect of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2, in vitro and in vivo, on neurotensin metabolism in the central nervous system was examined. 4. Pro-Phe-psi(PO2CHH2)-Leu-Pro-NH2 dose-dependently inhibited the formation of neurotensin 1-10 and concomittantly protected neurotensin from degradation by primary cultured neurones from mouse embryos. 5. Intracerebroventricular administration of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 significantly potentiated the neurotensin-induced antinociception of mice in the hot plate test. 6. Altogether, our study has established Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 as a fully selective and highly potent inhibitor of endopeptidase 3.4.24.16 and demonstrates, for the first time, the contribution of this enzyme in the central metabolism of neurotensin.     

Expression and estrogen regulation of the HEM45 MRNA in human tumor lines and in the rat uterus

The combination of manual and automated extraction procedures using low sample volumes (5-50 ml) with large-volume oncolumn injection (LVI) (200 microliters) in capillary gas chromatography with flame photometric detection (GC-FPD) has allowed the determination of 16 organophosphorus pesticides in clean water samples at the low ng l-1 level with an important simplification in the sample preparation step. A simple and fast offline liquid-liquid microextraction procedure (2-5 ml water/l ml methyl tert.-butyl ether) has been applied to spiked groundwater samples (containing 0.5 ng of each pesticide) with good recoveries (over 80%) and precision (better than 10%), giving detection limits between 5 and 100 ng l-1 using 200 microliters injections in the GC-FPD system. The application of an inline automated liquid-liquid microextraction-LVI-GC procedure (2 ml water/2 ml methyl tert.-butyl ether: injection of 200 microliters in GC-FPD) using the autosampler ASPEC XL led to lower recoveries (> 50%) as a result of the low efficiency for mixing organic and aqueous phases, although with very satisfactory coefficients of variation (lower than 7%) and detection limits between 20 and 200 ng l-1. Manual and automated solid-phase extraction procedures using the well known C18 cartridges and the new Oasis HLB have been applied to groundwater samples (5-50 ml) spiked with 1 ng of each pesticide. Results obtained for both the manual and the automated procedures were satisfactory (recoveries over 80%) and the limits of detection for 50 ml sample volume ranged from 1 to 6 ng l-1.