Improving the accuracy of the FMO binding affinity prediction of ligand-receptor complexes containing metals.

Polarization and charge transfer strongly characterize the ligand-receptor interaction when metal atoms are present, as for the Au(I)-biscarbene/DNA G-quadruplex complexes. In a previous work (J Comput Aided Mol Des2022, 36, 851-866) we used the ab initio FMO2 method at the RI-MP2/6-31G* level of theory with the PCM [1] solvation approach to calculate the binding energy (ΔEFMO) of two Au(I)-biscarbene derivatives, [Au(9-methylcaffein-8-ylidene)2]+ and [Au(1,3-dimethylbenzimidazole-2-ylidene)2]+, able to interact with DNA G-quadruplex motif. We found that ΔEFMO and ligand-receptor pair interaction energies (EINT) show very large negative values making the direct comparison with experimental data difficult and related this issue to the overestimation of the embedded charge transfer energy between fragments containing metal atoms. In this work, to improve the accuracy of the FMO method for predicting the binding affinity of metal-based ligands interacting with DNA G-quadruplex (Gq), we assess the effect of the following computational features: (i) the electron correlation, considering the Hartree-Fock (HF) and a post-HF method, namely RI-MP2; (ii) the two (FMO2) and three-body (FMO3) approaches; (iii) the basis set size (polarization functions and double-ζ vs. triple-ζ) and (iv) the embedding electrostatic potential (ESP). Moreover, the partial screening method was systematically adopted to simulate the solvent screening effect for each calculation. We found that the use of the ESP computed using the screened point charges for all atoms (ESP-SPTC) has a critical impact on the accuracy of both ΔEFMO and EINT, eliminating the overestimation of charge transfer energy and leading to energy values with magnitude comparable with typical experimental binding energies. With this computational approach, EINT values describe the binding efficiency of metal-based binders to DNA Gq more accurately than ΔEFMO. Therefore, to study the binding process of metal containing systems with the FMO method, the adoption of partial screening solvent method combined with ESP-SPCT should be considered. This computational protocol is suggested for FMO calculations on biological systems containing metals, especially when the adoption of the default ESP treatment leads to questionable results.

Non-invasive cardiovascular assessment of pregnancy in healthy female cats.

INTRODUCTION: The aim of this study was to evaluate cardiac morphology, indices of systolic and diastolic function, systolic blood pressure (SBP), and uterine artery resistance index (RI) during normal feline pregnancy. ANIMALS; MATERIALS AND METHODS: Twenty domestic short-hair pregnant queens, weighing 2.4-3.9 kg. Animals were assessed by two-dimensional, M-mode, and Doppler echocardiography, non-invasive SBP measurement, and uterine Doppler ultrasound every 10 days from mating to parturition. RESULTS: Interventricular septal thickness at end-diastole and end-systole, left ventricular internal dimension at end-diastole, left ventricular free wall thickness at end-diastole and end-systole, fractional shortening, stroke volume, cardiac output (CO), heart rate, and peak velocities of early and late diastolic transmitral flow increased during the second half of gestation, while SBP and RI decreased during the same period (p < 0.01). DISCUSSION AND CONCLUSIONS: Cardiac structure and function varied during normal pregnancy in these queens. Eccentric hypertrophy, increased indices of systolic function, and increased measurements of CO seem to be the consequences of the hemodynamic modifications occurring during pregnancy. The assessment of maternal cardiovascular function may prove a useful screening tool to detect pregnancy complications in feline species.

Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes.

In vitro models of differing macrophage functions are useful since human monocyte-derived macrophages are short-lived, finite and vary from donor to donor. Published protocols using the promonocytic cell line THP-1 have tended to result in cells that closely resemble classically-activated macrophages, differentiated in IFNγ and LPS. However, no protocol, to date, has fully recapitulated polarization of THP-1 to the M(IL-4) or M(IL-10) macrophage phenotypes seen when human monocyte-derived macrophages are exposed to each cytokine. Here we present protocols that can be used to prepare M(IL-4) polarized THP-1 that transcribe CCL17, CCL26, CD200R and MRC1 and M(IL-10) cells which transcribe CD163, C1QA and SEPP1. We show that the inhibitory Fcγ Receptor IIb is preferentially expressed on the surface of M(IL-4) cells, altering the balance of activating to inhibitory Fcγ Receptors. Adoption of standardized experimental conditions for macrophage polarization will make it easier to compare downstream effector functions of different macrophage polarization states, where the impact of PMA exposure is minimized and rest periods and cytokine exposure have been optimized.

Reference range of gestational uterine artery resistance index in small canine breeds.

The objectives of this study were to evaluate the uterine artery blood flow during the second half of normal pregnancy in small breed dogs and to determine the RI reference values of this artery in these bitches. Fifty-two, 1.5-6.5 kg, purebred healthy pregnant bitches were included in this study. Doppler ultrasonographic examinations of the uterus were performed every 10 days from Day 30 of the estrous cycle to parturition. Peak systolic velocity (PSV) and end diastolic velocity (EDV) of the uterine artery were measured while RI [(PSV-EDV)/PSV] was automatically calculated. Values of PSV, EDV and RI were analyzed by repeated measures ANOVA followed by Tukey test using age, body weight, litter size and parity as covariates. Resistance index reference ranges were constructed by regressing this index on gestational age. Equation of the polynomial regression curves was used to calculate the mean, 95th and 5th centiles for each gestational age (GA). A gradual increase of PSV (P < 0.01) and EDV (P < 0.01) and a decrease in RI (P < 0.01) were found throughout the study period. PSV and EDV were influenced by litter size (P < 0.05), while RI was not. None of the Doppler parameters were influenced by age, body weight or parity. A second-degree polynomial described the relationship between RI of uterine artery and GA in the different time points: [RI = 0.993 + 8.4x10-5 x GA2- 0.0117 x GA]. It is concluded that uterine artery blood flow progressively increased throughout normal pregnancy in small breed bitches. In addition, reference ranges of uterine artery RI were 0.64-0.79, 0.57 to 0.75, 0.53 to 0.71 and 0.52 to 0.66 on Days 30, 40, 50 and 60 of gestation, respectively.

Anti-inflammatory effects of essential oils from Mangifera indica.

Mangifera indica is widely found in Brazil, and its leaves are used as an anti-inflammatory agent in folk medicine. The aim of this study is to perform composition analysis of essential oils from the M. indica varieties, espada (EOMIL1) and coração de boi (EOMIL2), and confirm their anti-inflammatory properties. Twenty-three volatile compounds were identified via gas chromatography-mass spectrometry (GC-MS) in two essential oils from the leaves. Paw edema and myeloperoxidase (MPO) activity were evaluated using the carrageenan-induced paw model, while leukocyte migration was analyzed using the pleurisy model. At oral doses of 100 and 300 mg/kg, the essential oils significantly reduced edema formation and the increase in MPO activity induced by carrageenan in rat paws. For a dose of 300 mg/kg EOMIL1, 62 ± 8% inhibition of edema was observed, while EOMIL2 led to 51 ± 7% inhibition of edema. At a dose of 100 mg/kg, the inhibition was 54 ± 9% for EOMIL1 and 37 ± 7% for EOMIL2. EOMIL1 and EOMIL2 significantly reduced MPO activity at doses of 100 mg/kg (47 ± 5 and 23 ± 8%, respectively) and 300 mg/kg (50 ± 9 and 31 ± 7%, respectively). In the pleurisy model, inhibitions were also observed for EOMIL1 and EOMIL2 in the leukocyte migration test. The results of the present study show that essential oils from M. indica differ in chemical composition and anti-inflammatory activity in rats.

Electrocardiographic changes in normal and abnormal canine pregnancy.

The aim of this study was to describe the canine electrocardiographic changes in the course of normal and abnormal pregnancy. Twenty-three Brucellosis-negative pregnant bitches were retrospectively classified as normal (n = 12) or abnormal (n = 11). A control group of non-pregnant dioestrous bitches (n = 10) was also included. Normal pregnant females delivered healthy puppies at term while abnormal animals interrupted their pregnancy between days 52-60 (from estimated luteinizing hormone peak) or presented perinatal litter death higher than 60%. All the bitches were electrocardiographically evaluated every 10 days from day 0 to day 65 of the oestrous cycle, to parturition or abortion. Percentage heart rate change increased 31.3% from day 40 to 60 in normal gestation while it decreased -1.8% in dioestrous bitches, although it did not change in the abnormal group (p < 0.01). In the abnormal pregnant group but not in the others, percentage QRSa change fell to -34% on day 60 (p < 0.01). At the same time point, percentage QRSd change was 6.2% vs -4.9% in normal gestations and dioestrous animals, respectively (p < 0.05). Corrected QT interval augmented from day 40 onwards up to 9.9% and 4.3% in the normal pregnant and dioestrous groups, respectively, while it remained unchanged in abnormal gestations (p < 0.05). It is concluded that during normal canine pregnancy, some electrocardiographic parameters begin changing from day 40 onwards, and that pathological gestations differ from normality from day 30. The use of electrocardiography in canine obstetrics might contribute to identify abnormal outcomes before they become clinically evident.

Atom-bond pairwise additive representation for cation-benzene potential energy surfaces: An ab initio validation study.

The achievement of extensive and meaningful molecular dynamics simulations requires both the detailed knowledge of the basic features of the intermolecular interaction and the representation of the involved potential energy surface in a simple, natural and analytical form. This double request stimulated us to extend to ion-molecule systems a semiempirical method previously introduced for the description of weakly interacting atom-molecule aggregates and formulated in terms of atomic species-molecular bond interaction additivity. The method is here applied to the investigation of the prototypical M(+)-C6H6 systems (M = Li, Na, K, Rb and Cs) and some of its predictions are tested against accurate ab initio calculations. Such calculations have been performed by employing the MP2 method and large basis sets, privileging the description of the metal atoms. The agreement between potential energy scans semiempirically obtained and ab initio results is good for all the investigated geometries, thus showing that the adopted representation is in general able to reproduce all the main features of the potential energy surface for these systems. The role of the various noncovalent interaction components, as a function of the geometry and of the intermolecular distance in the M(+)-C6H6 complexes, is also investigated for a more detailed assessment of the results of the semiempirical method.

Lipomatosis pélvica: informe de un caso y revisión bibliográfica / Pelvic lipomatosis: case report and bibliography review

La prostatectomía radical está ampliamente difundida como tratamiento en el cáncer de próstata temprano. Pese a esto no queda claro su posible utilidad. Para responder a este interrogante realizamos un estu-dio randomizado. Métodos: Entre octubre de 1989 y febrero de 1996, 95 hombres con diagnóstico reciente de cáncer de próstata estadios 1 Ib, lie, o T2 fueron randomizados entre prostatectomía radical y control. Se realizó el seguimiento completo durante el ano 2000. El objetivo primario fue determinar la mortalidad por cáncer de próstata y secundaria la mortalidad gene-ral, la sobrevida libre de metástasis v la progresión local. Durante un promedio de seguimiento de 6,2 años, 62 hombres en el grupo de control y 53 en el de prostatectomía radi-ca] tallecieron. La muerte debida al cáncer de próstata ocurrió en 31 de los 348 pacientes del grupo control (8, 9 por ciento) y en 16 de los 347 asignados a prostatectomía radical (4,6 por ciento). La muerte relacionada con otras causas sobrevino en'31 de los 348 pacientes del grupo control (8,9 por ciento) y en 37 de los 347 pacientes asignados a prostatectomía radical (10,6por ciento) Los pacientes destinados a cirugía tuvieron un riesgo relativo menor para metástasis a distancia que los pacientes en el grupo control Conclusiones: En este estudio randomizado, la prostatectomía radical disminuyó significativamente la muerte asociada con la enfermedad, pero no presentó mayor diferencia con el grupo control en términos de sobrevida general.

A structural and theoretical analysis of transition metalloporphodimethenes and their relationship with metalloporphyrins.

The paper reports the synthesis of the first-row transition metal hexaethylporphodimethene derivatives [(Et6N4)M] [M=Mn, 3; M=Co, 5; M = Cu, 7] on a multigram scale, which makes them easily available for reactivity studies. After synthesis they were converted into the corresponding five-coordinate [(Et6,N4)M(L)] [M = Mn, L =THF, 8; M =Co, L = Py, 9] and six-coordinate [(Et6,N4)M(L)2] [M = Mn, L = THF, 10; M = Mn, L = Py, 11] derivatives. The compounds mentioned above and those recently reported, namely the iron and nickel derivatives 4, 6, 12, and 13, permit the presentation of the first coherent report on the structural, optical, magnetic, and electronic characteristics of the first-row transition metal porphodimethene derivatives. The experimental results, coupled with a detailed theoretical analysis (Density Functional Theory, DFT), give the appropriate background for future development of the porphodimethene skeleton, which paves the way from porphyrinogen to porphyrins. In addition, this report, encompassing the entire first row of transition metal ion porphodimethenes, allows a valuable comparison to be made with the corresponding metallated porphyrins, thus establishing the peculiar differences in terms of structural and electronic properties and potential reactivity.

Reduction of dioxygen by a dimanganese unit bonded inside a cavity provided by a pyrrole-based dinucleating ligand.

A novel class of dinucleating ligands has been introduced into manganese chemistry to study the reactivity of this metal towards dioxygen under strictly controlled conditions. Such N4 ligands combine some of the major peculiarities of tetradentate Schiff bases and the porphyrin skeleton. They are derived from the condensation between 2-pyrrolaldehyde and ethylenediamine or o-phenylenediamine, leading to pyrenH2 (LH2, 1), pyrophenH2 (L'H2, 2) and Me2pyrophenH2, (L"H2, 3), respectively. Their metallation with [Mn3-(Mes)6] (Mes = 2,4,6-trimethylphenyl) led to [Mn2L2] (4), [MnL'(thf)2] (5) and [MnL"(thf)2] (6). Complex 4 displays a double-stranded helical structure, while 5 and 6 are mononuclear complexes containing hexacoordinated metals. Regardless of their structure, complexes 5 and 6 behave in a similar manner to 4 in their reaction with dioxygen, namely, as a dimetallic unit inside a cavity defined by two dinucleating ligands. These reactions led to dinuclear MnIII/MnIV oxo-hydroxo derivatives, [Mn2L2(mu-O)(mu-OH)] (7), [Mn2L'2(mu-O)(mu-OH)] (8) and [Mn2L"2(mu-O)(mu-OH)] (9), in which the two Mn ions are strongly antiferromagnetically coupled [J = -53 (7), J = -64 (8), J = -60 cm(-1) (9)]. The crystal structure of 7 could only be solved with synchrotron radiation as the crystals diffracted very poorly and suffered from twisting and disorder. The formation of 7-9 has been proposed to occur through the formation of an intermediate dinuclear hydroperoxo species.

Density functional study of tetraphenolate and calix[4]arene complexes of early transition metals.

Density functional calculations have been performed on some calix[4]arenes complexes of early transition metals. Particular emphasis has been placed on the comparison of the main properties of these metal complexes with the analogous metal complexes based on four monodentate phenolate ligands to study the effect of the geometrical constraints imposed by the calixarenes framework on the electronic structure. The results show that the most stable geometry of titanium and molybdenum tetraphenolates is pseudotetrahedral (slightly flattened for molybdenum) and that the distortion to a square planar coordination requires, respectively, 52.0 and 21.5 kcal x mol(-1). However, a significant energy decrease is found when the four phenolate groups are bent in the same hemisphere, reproducing the calix[4]arene geometry. Such a coordination determines the energy decrease of the unoccupied metal d orbitals of sigma and pi symmetry, which leads to an increase of the electron-accepting properties of these metal fragments.

The binding ability of iron bonded to porphodimethene: structural, magnetic, and electronic relationship to iron porphyrin complexes.

The availability of the parent compound, meso-hexaethylporphodimetheneiron(II), [(Et6N4)Fe] (2), of this report results from a novel synthetic methodology that makes [Et6N4Li2] (1) easily available. The major focus is on how the axial positions, which are the key reactive sites in metalloporphyrins, and the electronic configuration of the metal can be affected by the breakdown of the aromaticity of the porphyrin skeleton and by the nonplanar conformation of the ligand. DFT calculations indicate a 3B1(dz2)1(dyz)1 ground state for 2 versus the 3A2(dxz)1(dyz)1 ground state in the porphyrin analogue. The intermediate-spin state (S = 1) of 2 changed drastically upon addition of one or two axial ligands, as hexacoordination is preferred by iron(II). The hexacoordinate complexes [(Et6N4)Fe(L)(L')] (L = L' = THF, 3; L = L' = Py, 4; L = PhNO, L' = Py, 14) have been isolated and structurally characterized. Strong-field ligands lead to a low-spin diamagnetic state for iron(II), namely for complexes 4-7, 9, and 14, whereas 3 is a typical d6 high-spin complex, as is the pentacoordinate [(Et6N4)Fe(CN)]Bu4N (8). The structural analysis showed common features for 6, 7, 9, and 14: i) a small displacement of the metal from the N4 plane, and ii) an N4 cavity, larger than that in the corresponding porphyrins, affecting the Fe-N bond lengths. The 1H NMR spectrum is quite diagnostic of the two-fold symmetry in the diamagnetic hexacoordinate complexes, revealing either a D2h or a C2v symmetry. The CO stretching frequency (1951 cm(-1)) in complex 6 probes the good electron density at the metal. The one-electron oxidation of 2 led to pentacoordinate iron(III) derivatives [(Et6N4)Fe(Cl)] (10), [(Et6N4)2Fe2(mu-O)] (11), and [(Et6N4)2Fe2(mu-p-OC6H4-O)] (12). Complex 10 is a typical high-spin iron(III) (5.85muB at 298 K), while 11 and 12 behave as antiferromagnetic coupled iron(III) (J = -9.4cm(-1), 12, and J = -115cm(-1), 11). In complexes 10, 11, and 12 iron is sitting in a quite distorted square pyramidal geometry, in which the ligand displays a very distorted roof conformation with different degrees of ruffling. Distinctive structural and magnetic features have been found for the nitrosyl derivative [(Et6N4)Fe-NO], which has a low-spin state (S = 1/2) and the following structural parameters: Fe-N-O, 147.3(2) degrees; Fe-N, 1.708(2) A; N-O, 1.172(3) A. A comparative structural, magnetic, and theoretical analysis of the compounds listed above has been made with the analogous porphyrin derivatives. The detailed structural investigation has been mapped through the X-ray analysis of 2, 7, 8, 9, 11, 13, and 14.

Binding and redox properties of iron(II) bonded to an oxo surface modeled by calix[4]arene.

The syntheses of the parent compounds [(p-Bu(t)-calix[4]-(O)2(OR)2)Fe-L] [R = Me, L = THF, 5; R = Bu(n), L = THF, 6; R = PhCH2, L = THF, 7; R = SiMe3, L = none, 8] have been performed by reacting the protonated form of the dialkylcalix[4]arene with [Fe2Mes4] [Mes = 2,4,6-Me3C6H2]. All of them undergo one-electron oxidative functionalization. By use of different oxidizing agents, the following iron(III) derivatives have been obtained: [(p-Bu(t)-calix[4]-(O)2(OR)2)Fe-X] [X = Cl, R = Me, 9; X = I, R = Me, 10] and [(p-Bu(t)-calix[4]-(O)2(OR)2)2Fe2(mu-X] [X = O, R = Me, 11; X = O, R = Bu(n), 12; X = S, R = Me, 13], 9 and 10 being particularly appropriate for a further functionalization of the metal. The last three display typical antiferromagnetic behavior [J = -78.6 cm-1, 11; J = -64.1 cm-1, 13]. In the case of 7 and 8, the reaction with O2 led to the dealkylation of one of the alkoxo groups, with the formation of a dimeric iron(III) derivative ([mu-p-Bu(t)-calix[4]-(O)3(OR))2Fe2] [R = PhCH2, 14; R = SiMe3, 15] [J = -9.8 cm-1]. The reaction of the parent compounds with ButNC and diazoalkanes led to the formation of [Fe=C] functionalities supported by a calix[4]arene oxo surface. The following compounds have been isolated and characterized: ([p-Bu(t)-calix[4]-(O)2(OR)2)Fe=CNBut] [R = SiMe3, 16, nu CN = 2175 cm-1], ([p-Bu(t)-calix[4]-(O)2(OR)2)Fe=CPh2] [R = Me, 17; R = PhCH2, 18; R = SiMe3, 19]. The three carbene complexes 17-19 display quite an unusual high-spin state, which is a consequence of the formation of a weak pi interaction between the metal and the carbene carbon, as confirmed by the extended Hückel calculations. The carbene functionality has been removed from the iron center in the reaction with O2 and HCl. The proposed structures have been supported by X-ray analyses of complexes 8, 9, 12, 14, 16, 17, and 19.

Oligomerization of early transition metal [M(OH)3]2(mu-C2) acetylides toward the formation of [(OH)3MC]n (n = 4, 6) metalla carbides: a theoretical study by density functional theory.

Density functional calculations were performed on a series of M(OH3)-substituted (M = Ti,V) cyclopolyenes as simple models is metal carbides. We studied the oligomerization of the metal acetylide complexes [MLn]2 (mu-C2) as a possible precursor of these [(OH)3MC]n (n = 4,6) hypothetical species. Special emphasis was placed on the comparison of the main properties of these metal substituted cyclopolyenes with those of the corresponding cyclopolyenes in an attempt to study the effects of the metal substituents on the organic C4 and C6 cyclic moieties. Whereas for the titanium species, the pi system of the polyene moiety is slightly perturbed, the electronic structures and molecular geometries found for the vanadium species suggest a metalla radialene nature for these compounds, with the endocyclic conjugation of carbon-carbon double bonds replaced by an exocyclic arrangement of carbon-metal double bonds.

Cabergoline in acromegaly: a renewed role for dopamine agonist treatment?

OBJECTIVE AND DESIGN: Eighteen active acromegalics entered a prospective open study with cabergoline (CAB), a dopaminergic drug much more potent than bromocriptine (Br). METHODS: CAB was administered for 6 months at doses ranging between 0.5 mg twice weekly and 0.5 mg/day. Clinical-anamnestic characteristics of the patients were: (i) sensitivity to dopamine agonist drugs (10 patients); (ii) resistance to somatostatin analogs (SAs) (8 patients): (iii) intolerance to SA (3 patients). In 2 patients marked hyperprolactinemia was present. RESULTS: Basal GH was 6.6 microg/l (2.2-50) (median (range)), and on treatment it was 3.5 microg/l (1.2-34) (P=0.013). The corresponding IGF-I values were 720 microg/l (410-1438) and 375 microg/l (167-1260) respectively (P=0.00001). Individual GH levels decreased below 2 microg/l in 5 patients, and between 2 and 5 microg/l in another 5 patients. IGF-I levels were suppressed below 50% of baseline in 8 patients and normal age-adjusted IGF-I values were reached in 5 patients (27% of the series). The retrospective comparison with previous chronic treatment with Br in the 10 suitable patients showed a greater effectiveness of CAB (IGF-I decrease on CAB treatment, 46.8%, on Br treatment, 31%, P=0.02). Adenoma shrank in the 3 patients whose pituitary imaging was repeated during CAB. CONCLUSIONS: These results envisage that CAB may represent a worthy therapeutic tool in acromegalic patients, inducing a degree of IGF-I and GH suppression comparable to SAs, administered by the oral route and much less expensive.

Effects of tamoxifen on GH and IGF-I levels in acromegaly.

Tamoxifen (TAM), a non steroid partially competitive antagonist to the estrogen receptors, has been reported to decrease plasma GH and IGF-I levels both in vitro and in vivo. These data prompted us to evaluate GH and IGF-I changes in acromegaly after acute and chronic TAM administration. Nineteen acromegalic patients (6 M, 13 F, aged 30-70 years) were studied in a prospective open study. Acute TAM test (20 mg po) did not induce any significant change in GH and IGF-I levels. Chronic TAM treatment (20 mg/day for a month and 40 mg/day for another month) induced a transient increase in GH levels (from 9 [3-139] micrograms/l [median, range] to 12 [3-188] micrograms/l, p = 0.0025) and a persistent decrease in IGF-I levels (from 785 [500-1200] micrograms/l to 553 [209-1420] micrograms/l, p = 0.0034). Individual IGF-I values decreased in 13 patients and reached the normal range in 4 of them. At TAM withdrawal hormonal levels increased up to pretreatment values. There was no correlation between GH and IGF-I changes and results were not influenced by age, sex or gonadal status. In this setting it is likely that the observed decrease in plasma IGF-I levels is dependent on TAM activity at the hepatic level.

Experience with hair testing in the clinical biochemistry laboratory of Ca' Granda Niguarda Hospital, Milan, Italy.

In our laboratory, analysis of human hair for drugs of abuse detection was first performed in 1980. In the last 10 years we have processed about 2000 subjects/year ('living subjects' only). In the last 3 years we have also introduced hair analysis of cocaine: at first only in clinical applications, but for the last 2 years this analysis is now routine. Our application of hair analysis includes: clinical toxicology, medico-legal and administrative agencies. Requests come for example from several Committees for Driving Licenses, Addiction Treatment Centers and Legal Authorities. Hair samples are currently collected from the occipital area at the back of the head, which appears to show less variability in hair growth rate. At present we perform hair analysis using highly sensitive radioimmunoassay screening methods for the detection of parent drug and/or metabolites. All positive cases of cocaine and opiates abuse are confirmed by gas chromatography-mass spectrometry in electron impact or chemical ionization mode. Positive cases for opiates are also analysed using a specific morphine radioimmunoassay kit. Data show that, when the opiates/morphine ratio is higher than 6, we are dealing with consumption of codeine and/or dihydrocodeine. In our routine work last year there were 177 (263 samples) positive opiates subjects out of 2244 patients; positive cocaine subjects were 290 (362 samples) out of 2001 patients. Guidelines for hair analysis in Lombardia have been established based on the experience of our laboratory. Furthermore it will be possible to apply a unique protocol for all Committees for Driving Licenses, involving hair testing in addition to urine assay.