Characterization of a rare triterpenoid and minor phenolic compounds in the root bark of Anisophyllea dichostyla R. Br.

Anisophyllea dichostyla R. Br. (Anisophylleaceae), is a small shrub which grows widely in regions of the Democratic Republic of Congo (DRC) where its root barks are used in folk medicine for the treatment of many debilitating diseases. In a previous work [Khallouki, F., Haubner, R., Hull, W.E., Erben, G., Spiegelhalder, B., Bartsch, H., Owen, R.W., 2007. Isolation, purification and identification of ellagic acid derivatives, catechins and procyanidins from the root barks of Anisophyllea dichostyla R. Br. Food and Chemical Toxicology 45, 472-485] on this species, an appreciable number (16) of phenolic antioxidants (3.32 g/kg) such as ellagitannins (27%) and polyhydroxyflavan-3-ols (catechins and procyanidins; 73%) were isolated and identified. Two fractions, as well as containing minor phenolic compounds also showed evidence of a secondary plant substance similar to a triterpenoid. Following purification of the triterpenoid by semi-preparative HPLC, and recrystallization, the structure was elucidated as bryonolic acid as evinced by comprehensive spectroscopic analyses including (1)H and (13)C NMR, DEPT, COSY, ROESY, HMQC, HMBC, HPLC-ESI-MS and GC-MS experiments. Bryonolic acid, which is extremely rare in nature, is therefore reported in the family Anisophylleaceae for the first time. Furthermore, the following minor phenolic compounds namely tyrosol, 2-(3-methoxy, 4-hydroxyphenyl)-ethanol, vanillin, syringaldehyde, vanillic acid, syringic acid, gallic acid and ferulic acid were also identified by GC-MS in this species for the first time.

Isolation, purification and identification of ellagic acid derivatives, catechins, and procyanidins from the root bark of Anisophyllea dichostyla R. Br.

The root bark of Anisophyllea dichostyla R. Br. is traditionally used in the Democratic Republic Congo for the treatment of several conditions such as anorexia, fatigue and intestinal infections. We have identified and quantitated several polyphenol antioxidants in the methanol extract of the root bark (120g). The polyphenol content (3.32g/kg) was predominantly ellagitannins (25%) and polyhydroxyflavan-3-ols (catechins and procyanidins, 75%) with 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside and (-)-epicatechin as the major species in each class. These two compounds and the following species were identified unequivocally by NMR spectroscopy: (+)-catechin, (-)-epicatechin 3-O-gallate, 3-O-methyl ellagic acid, 3,3'-di-O-methyl ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside, and 3'-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. The following additional compounds were purified by semi-preparative HPLC and tentatively identified on the basis of UV spectra, HPLC-ESI-MS and nano-ESI-MS-MS: (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), epicatechin-(4beta-->8)-epicatechin (procyanidin B(2)), an (epi)catechin trimer, 3-O-methyl ellagic acid 4-O-beta-d-glucopyranoside, (-)-epicatechin 3-O-vanillate, 3,4-methylenedioxo ellagic acid 4'-O- beta-d-glucopyranoside, and 3,3'-di-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. Fractionation of the raw extract by column chromatography on silicic acid yielded 10 fractions. In the hypoxanthine/xanthine oxidase antioxidant assay system, CC-9 which contained a range of polyphenols dominated by (-)-epicatechin-O-gallate proved to be the most potent antioxidant fraction (IC(50)=52 micro g/mL) in terms of ROS scavenging. In terms of XO inhibition CC-8, dominated by (epi)catechin trimer and which also contained appreciable amounts of 3'-O-methyl ellagic acid 4'-O-beta-d-xylopyranoside, as well as the catechins (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), and (-)-epicatechin 3-O-gallate, proved to be the most potent (IC(50)=36 micro g/mL).

Analysis of enterolignan glucuronides in serum and urine by HPLC-ESI-MS.

A method involving the coupling of high-performance liquid chromatography with electrospray ionisation mass spectrometry (HPLC-ESI-MS) for the quantitative determination of the mammalian lignans enterolactone and enterodiol in human blood and urine has been developed. In contrast to techniques previously published, the method allows direct measurement of free enterolignans as well as their monoglucuronide conjugates in human biofluids with minimal sample preparation. Thereby the method is suitable for large-scale intervention, case-control and epidemiologic studies. Comprehensive, high-precision (1)H and (13)C nuclear magnetic resonance data (CD3OD as solvent) obtained at 11.7 T in combination with polarimetric data show that the major form of lignan precursor in the linseeds used is (-)-secoisolariciresinol diglucoside ((2R,3R)-2,3-bis(4'-hydroxy-3'-methoxy-benzyl)-1,4-butanediyl-bis-beta-d-glucopyranoside) which is transformed by human intestinal bacteria into (+)-enterodiol and (+)-enterolactone. However, these metabolites are mono-glucuronidated after absorption and are detected as (-)-enterodiol 3'-beta-d-glucuronide=(2R,3R)-2-(3'-O-(beta-d-glucopyranosyluronic acid)benzyl)-3-(3''-hydroxybenzyl)-butane-1,4,diol and (-)-enterolactone 3'-beta-d-glucuronide=(2R,3R)-2-(3'-O-(beta-d-glucopyranosyluronic acid)benzyl)-3-(3''-hydroxybenzyl)-beta-butyrolactone in blood and urine.

Isolation and structure elucidation of phenolic antioxidants from Tamarind (Tamarindus indica L.) seeds and pericarp.

Although it is already known that Tamarind (Tamarindus indica L.) seeds contain phenolic substances, the individual components of the seeds have not been fully identified and quantitated, and in the case of Tamarind pericarp not reported. Therefore, major polyphenolic compounds were extracted using organic solvents and the metabolites were isolated by semi-preparative high performance liquid chromatography. Their structures were elucidated by liquid chromatography-electrospray-ionisation-mass spectrometry (LC-ESI-MS), nano-electrospray-ionisation mass spectrometry (ESI-MS), and where possible by gas chromatography-mass spectrometry (GC-MS) and 1H and 13C NMR. Quantitative analysis of polyphenolic compounds in Tamarind seeds and pericarp was conducted by analytical high performance liquid chromatography (HPLC), calculated against standard curves of authentic compounds. The yields of total phenolic compounds after Soxhlet extraction with methanol were 6.54 and 2.82 g/kg (dry weight) in the seeds and pericarp respectively. The profile (%) of polyphenolics in Tamarind pericarp was dominated by proanthcyanidins (73.4) in various forms (+)-catechin (2.0), procyanidin B2 (8.2), (-)-epicatechin (9.4), procyanidin trimer (11.3), procyanidin tetramer (22.2), procyanidin pentamer (11.6), procyanidin hexamer (12.8) along with taxifolin (7.4), apigenin (2.0), eriodictyol (6.9), luteolin (5.0) and naringenin (1.4) of total phenols, respectively. The content of Tamarind seeds comprised only procyanidins, represented (%) mainly by oligomeric procyanidin tetramer (30.2), procyanidin hexamer (23.8), procyanidin trimer (18.1), procyanidin pentamer (17.6) with lower amounts of procyanidin B2 (5.5) and (-)-epicatechin (4.8). Extraction of Tamarind pericarp and seeds using acetone:methanol:acetic acid gave only procyanidin oligomers, but in much higher yield and variety. The antioxidant capacities of the Soxhlet methanolic extracts were determined, and indicates that Tamarind may be an important source of cancer chemopreventive natural products in tropical regions.

Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays.

KTCTL-26 and KTCTL-2 are renal cell carcinoma (RCC) lines with high and low expression of P-170 glycoprotein, respectively. Inherent differences between the two cell lines in terms of phosphate metabolites and growth characteristics in culture were examined for possible association with multidrug resistance (MDR). Differences in response to drug treatment were investigated for 40 h incubations with various doses of vinblastine (VBL) alone or as cotreatments with various concentrations of the calcium antagonist diltiazem (DIL) and/or interferon-alpha (IFN-alpha). Treatment effects were quantitated using the MTT survival assay and 31P magnetic resonance spectroscopy (MRS) to determine phosphate metabolite profiles in intact cells. KTCTL-2 and KTCTL-26 cells exhibited significant inherent differences in phosphocholine, glycerophosphocholine, glycerophosphoethanolamine, and phosphocreatine levels. KTCTL-26 cells were more sensitive than KTCTL-2 to 0.011 mircroM VBL alone (87% vs. 102% survival) or to 0.011 microM BL + 10 microM DIL (55% vs. 80% survival). The latter treatment resulted in a significant decrease in the ratio of phosphocholine to glycerophosphocholine in KTCTL-26 cells but no significant changes in phosphate metabolites in KTCTL-2 cells. Metabolomic 31P MRS detects different metabolite profiles for RCC cell lines with different MDR phenotypes and may be useful for noninvasive characterization of tumors in a clinical setting.

Isolation and structure elucidation of the major individual polyphenols in carob fibre.

Although it is already known that carob fibre contains several classes of polyphenolic substances, a comprehensive analysis of these has not been conducted to date. Therefore, the major polyphenolic compounds were extracted with organic solvents, and, following fractionation by normal-phase column chromatography on silicic acid, their structures were elucidated by liquid-chromatography electrospray-ionisation mass spectrometry (LC-ESI), nano-electrospray-ionisation mass spectrometry (ESI-MS), and gas-chromatography mass spectrometry (GC-MS). In addition, complete 1H and 13C NMR assignments were obtained for the isolated gallotannins 1,6-di-, 1,2,6-tri- and 1,2,3,6-tetra-O-galloyl-beta-D-glucose. Carob fibre was found to contain a rich variety of phenolic antioxidants. A total of 24 polyphenol compounds were identified with a yield of 3.94 g/kg (dry weight). The profile was dominated by gallic acid in various forms: free gallic acid (42% of polyphenols by weight), gallotannins (29%), and methyl gallate (1%), while simple phenols, mainly cinnamic acid, made up about 2% of the total. Flavonoids represented 26% of the polyphenols, and the major components were identified as the glycosides myricetin- and quercetin-3-O-alpha-L-rhamnoside (ca. 9% and 10%, respectively). These data indicate that carob fibre is rich in both amount and variety of phenolic antioxidant substances, and its inclusion in the diet may have chemopreventive properties.

Correlation between 5-fluorouracil metabolism and treatment response in two variants of C26 murine colon carcinoma.

Following an i.p. dose of 150 mg x kg(-1) 5-fluorouracil (5-FU), drug uptake and metabolism over a 2-h period were studied by in vivo (19)F magnetic resonance spectroscopy (MRS) for the murine colon carcinoma lines C26-B (5-FU-insensitive; n=11) and C26-10 (5-FU-sensitive; n=15) implanted s.c. in Balb/C mice. Time courses for tumour growth, intracellular levels of FdUMP, thymidylate synthase (TS) activity, and 5-FU in RNA were also determined, and the effects of a 9.5-min period of carbogen breathing, starting 1 min before drug administration, on MRS-detected 5-FU metabolism and tumour growth curves were examined. Both tumour variants generated MRS-detectable 5-FU nucleotides and showed similar initial growth inhibition after treatment. However, the growth rate of C26-B tumours returned to normal, while the sensitive C26-10 tumours, which produced larger fluoronucleotide pools, still showed moderate growth inhibition. Carbogen breathing did not significantly influence 5-FU uptake or fluoronucleotide production but did significantly enhance growth inhibition in C26-10 tumours. While both tumour variants exhibited incorporation of 5-FU into RNA and inhibition of TS via FdUMP, clearance of 5-FU from RNA and recovery of TS activity were greater for the insensitive C26-B line, indicating that these processes, in addition to 5-FU uptake and metabolism, may be important determinants of drug sensitivity and treatment response.

Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes.

Because olives represent an important component of the Mediterranean diet, it is necessary to establish unequivocal identification and quantitation of the major potential antioxidant phenolic compounds they contain. The major phenolic antioxidants in two types of brined olives were isolated and purified by semi-preparative high performance liquid chromatography. Structural analysis was conducted using UV spectrophotometry, mass spectrometry and nuclear magnetic resonance spectroscopy. In particular, completely assigned 1H and 13C NMR data are presented and errors in literature data are corrected. The data show that tyrosol, hydroxytyrosol, 3-(3, 4-dihydroxyphenyl) propanoic acid (dihydrocaffeic acid), dihydro-p-coumaric acid (phloretic acid), the phenylpropanoid glucosides acteoside (verbascoside) and isoacteoside, along with the flavonoids luteolin and apigenin are major components of the phenolic fraction of brined black olives. Brined green olives contain only hydroxytyrosol and traces of other minor phenolics. Brined olives contain even higher concentrations of phenolic antioxidants than olive oil and may, therefore, be more important modulators of cancer chemopreventive activity.

Cytosine deaminase and thymidine kinase gene therapy in a Dunning rat prostate tumour model: absence of bystander effects and characterisation of 5-fluorocytosine metabolism with 19F-NMR spectroscopy.

The rat prostate tumour cell line R3327 AT-1 was transfected with a gene coding for a fusion protein comprised of cytosine deaminase (CD from E. coli) and thymidine kinase (TK from Herpes simplex virus, HSV-1). The resulting AT-1/CDglyTK cell line was sensitive to the prodrug 5-fluorocytosine (IC(50) = 78 microM, 96-h incubation) via CD and to ganciclovir (GCV, IC(50) = 1 microM, 96 h) via TK. Subcutaneous tumours generated from 100% CDglyTK(+) cells responded well to 5-FC therapy (500 mg/kg, i.p., 14 daily treatments, four out of seven animals in remission) and to GCV therapy (30 mg/kg, i.p., 14 daily treatments, five of six animals in remission). However, experiments with mixtures of CDglyTK(+) and CDglyTK(-) cells showed low levels of connexins (intercellular gap junctions) and no bystander effect for nontransfected cells using either 5-FC or GCV therapy. Furthermore, (19)F-NMR spectroscopy showed that incubation of cultured CDglyTK(+) cells with 774 microM 5-FC for 16 h resulted in the following intracellular concentrations: 5-FC = 314 microM, 5-FU = 52 microM, cytotoxic fluoronucleotides = 163 microM; extracellular 5-FU reached only 6.4 microM. Thus, in this model system intracellular trapping of 5-FU (slow export) contributes to the failure of the CD/5-FC bystander effect via an extracellular route.

Monosaccharide-linked inhibitors of O(6)-methylguanine-DNA methyltransferase (MGMT): synthesis, molecular modeling, and structure-activity relationships.

A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.

SP-D and GM-CSF regulate surfactant homeostasis via distinct mechanisms.

Both surfactant protein (SP) D and granulocyte-macrophage colony-stimulating factor (GM-CSF) influence pulmonary surfactant homeostasis, with the deficiency of either protein causing marked accumulation of surfactant phospholipids in lung tissues and in the alveoli. To assess whether the effects of each gene were mediated by distinct or shared mechanisms, surfactant homeostasis and lung morphology were assessed in 1) double-transgenic mice in which both SP-D and GM-CSF genes were ablated [SP-D(-/-),GM(-/-)] and 2) transgenic mice deficient in both SP-D and GM-CSF in which the expression of GM-CSF was increased in the lung. Saturated phosphatidylcholine (Sat PC) pool sizes were markedly increased in SP-D(-/-),GM(-/-) mice, with the effects of each gene deletion on surfactant Sat PC pool sizes being approximately additive. Expression of GM-CSF in lungs of SP-D(-/-),GM(-/-) mice corrected GM-CSF-dependent abnormalities in surfactant catabolism but did not correct lung pathology characteristic of SP-D deletion. In contrast to findings in GM(-/-) mice, degradation of [(3)H]dipalmitoylphosphatidylcholine by alveolar macrophages from the SP-D(-/-) mice was normal. The emphysema and foamy macrophage infiltrates characteristic of SP-D(-/-) mice were similar in the presence or absence of GM-CSF. Taken together, these findings demonstrate the distinct roles of SP-D and GM-CSF in the regulation of surfactant homeostasis and lung structure.

Surfactant protein deficiency in familial interstitial lung disease.

OBJECTIVE: To determine the contribution of surfactant protein abnormalities to the development of chronic lung injury in a familial form of interstitial lung disease. STUDY DESIGN: An 11-year-old girl, her sister, and their mother who were diagnosed with chronic interstitial lung disease underwent laboratory investigation of surfactant protein expression in bronchoalveolar lavage fluid and lung biopsy specimens. Nineteen patients with idiopathic pulmonary fibrosis and 9 patients who were investigated for pulmonary malignancy but who did not have interstitial lung disease served as control subjects. RESULTS: The 3 family members were found to have absent surfactant protein C (SP-C) and decreased levels of SP-A and SP-B in bronchoalveolar lavage fluid (BALF). Immunostaining for pulmonary surfactant proteins in lung biopsy specimens obtained from both children demonstrated a marked decrease of pro-SP-C in the alveolar epithelial cells but strong staining for pro-SP-B, SP-B, SP-A, and SP-D. No deviations from published surfactant protein B or C coding sequences were identified by DNA sequence analysis. All control subjects had a detectable level of SP-C in the BALF. CONCLUSION: The apparent absence of SP-C and a decrease in the levels of SP-A and SP-B are associated with familial interstitial lung disease.

Radioiodinated N-(2-diethylaminoethyl)benzamide derivatives with high melanoma uptake: structure-affinity relationships, metabolic fate, and intracellular localization.

Several radioiodinated N-(dialkylaminoalkyl)benzamides have been used for planar scintigraphy and single-photon emission computed tomography (SPECT) of melanoma metastases. In a quest for improved melanoma uptake and tissue selectivity, structure-activity studies for N-(2-diethylaminoethyl)benzamides with variation of phenyl substituents were performed using C57Bl/6 mice bearing B16 melanoma. Compounds 2 (4-amino-5-bromo-N-(2-diethylaminoethyl)-3-[(131)I]iodo-2-methoxybenz amide) and 6 (4-acetamido-N-(2-diethylaminoethyl)-5-[(131)I]iodo-2-methoxybenzamid e) showed at 6 h post iv injection, for example, melanoma uptake of 16.6 and 23.2% ID/g, respectively (mean values, n = 3). Uptake was 3-5 times higher (P < 0.01) than observed with benzamides known from the literature and was probably facilitated by the relatively slow urinary excretion of 2 or 6. In contrast, analogues lacking either the MeO, Ac, AcNH, or Br substituents exhibited reduced tumor uptake and high urinary excretion of radioactivity in various benzamide metabolites. Uptake of radioiodinated benzamides in B16 melanoma is not mediated by a specific mechanism such as sigma-receptor binding. 2 and 6 exhibited similar melanoma uptake values but quite different sigma(1)-receptor affinities of K(i) = 0.278 +/- 0.018 and 5.19 +/- 0.40 microM, respectively. Uptake studies with IMBA (N-(2-diethylaminoethyl)-3-[(131)I]iodo-4-methoxybenzamide) or BZA (N-(2-diethylaminoethyl)-4-[(131)I]iodobenzamide) showed that with increasing dose of unlabeled compound the measured uptake of label was unchanged (IMBA) or even enhanced (BZA) while receptor binding of label decreased. Differential and equilibrium density-gradient centrifugation revealed that most of the radioactivity from labeled IMBA was associated with fractions containing melanin granules. Thus, structure-activity studies indicate that blood clearance rates and metabolic stability are the main determinants for benzamide uptake in melanoma. The high uptake and slow clearance of 6 offer considerable potential for melanoma imaging in patients, and this compound may also prove to be useful for radionuclide therapy.

Identification of lignans as major components in the phenolic fraction of olive oil.

BACKGROUND: Because olive oil is an important component of the Mediterranean diet, it is necessary to establish unequivocal identification of the major potential antioxidant phenolic compounds it contains. METHODS: The major phenolic antioxidants in extra virgin olive oil were isolated and purified. Structural analysis was conducted using several spectroscopic techniques, including mass spectrometry and nuclear magnetic resonance (NMR). In particular, detailed (1)H and (13)C NMR data are presented, and several assignment errors in the literature are corrected. RESULTS: The data show for the first time that the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol are major components of the phenolic fraction of olive oils. These lignans, which are potent antioxidants, are absent in seed oils and virtually absent in refined virgin oils but are present at concentrations of up to 100 mg/kg (mean +/- SE, 41.53+/-3.93 mg/kg; range, 0.65-99.97 mg/kg) in extra virgin oils. As with the simple phenols and secoiridoids, there is considerable interoil variation in lignan concentrations. Foods containing high amounts of lignan precursors have been found to be protective against breast, colon, and prostate cancer. CONCLUSION: Lignans, as natural components of the diet, may be important modulators of cancer chemopreventive activity.

Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignansand squalene.

The aim of this study was to evaluate the phenolic antioxidant and squalene content in a range of olive and seed oils. A mean of 290 +/- 38 (SEM) mg squalene/100 g was detected. However, while there was a weak significant difference between extra virgin (424 +/- 21 mg/kg) and refined virgin (340 +/- 31 mg/100 g; P<0.05) olive oils, highly significant differences were evident between extra virgin olive oils (P<0.0001) refined virgin olive oils (P<0.0001) and seed oils (24 +/- 5 mg/100 g). While seed oils were devoid, on average, the olive oils contained 196 +/- 19 mg/kg total phenolics as judged by HPLC analysis, but the value for extra virgin (232 +/- 15 mg/kg) was significantly higher than that of refined virgin olive oil (62 +/- 12 mg/kg; P<0.0001). Appreciable quantities of simple phenols (hydroxytyrosol and tyrosol) were detected in olive oils, with significant differences between extravirgin (41.87 +/- 6.17) and refined virgin olive oils (4.72 +/- 215; P<0.01). The major linked phenols were secoiridoids and lignans. Although extra virgin contained higher concentrations of secoiridoids (27.72 +/- 6.84) than refined olive oils (9.30 +/- 3.81) this difference was not significant. On the other hand, the concentration of lignans was significantly higher (P<0.001) in extra virgin (41.53 +/- 3.93) compared to refined virgin olive oils (7.29 +/- 2.56). All classes of phenolics were shown to be potent antioxidants. In future epidemiologic studies, both the nature and source of olive oil consumed should be differentiated in ascertaining cancer risk.

The antioxidant/anticancer potential of phenolic compounds isolated from olive oil.

In our ongoing studies on the chemoprevention of cancer we have a particular interest in the health benefits of the Mediterranean diet, of which olive oil is a major component. Recent studies have shown that extravirgin olive oil contains an abundance of phenolic antioxidants including simple phenols (hydroxytyrosol, tyrosol), aldehydic secoiridoids, flavonoids and lignans (acetoxypinoresinol, pinoresinol). All of these phenolic substances are potent inhibitors of reactive oxygen species attack on, e.g. salicylic acid, 2-deoxyguanosine. Currently there is growing evidence that reactive oxygen species are involved in the aetiology of fat-related neoplasms such as cancer of the breast and colorectum. A plausible mechanism is a high intake of omega-6 polyunsaturated fatty acids which are especially prone to lipid peroxidation initiated and propagated by reactive oxygen species, leading to the formation (via alpha,beta-unsaturated aldehydes such as trans-4-hydroxy-2-nonenal) of highly pro-mutagenic exocyclic DNA adducts. Previous studies have shown that the colonic mucosa of cancer patients and those suffering from predisposing inflammatory conditions such as ulcerative colitis and Crohn's disease generates appreciably higher quantities of reactive oxygen species compared with normal tissue. We have extended these studies by developing accurate high performance liquid chromatography (HPLC) methods for the quantitation of reactive oxygen species generated by the faecal matrix. The data shows that the faecal matrix supports the generation of reactive oxygen species in abundance. As yet, there is a dearth of evidence linking this capacity to actual components of the diet which may influence the colorectal milieu. However, using the newly developed methodology we can demonstrate that the antioxidant phenolic compounds present in olive oil are potent inhibitors of free radical generation by the faecal matrix. This indicates that the study of the inter-relation between reactive oxygen species and dietary antioxidants is an area of great promise for elucidating mechanisms of colorectal carcinogenesis and possible future chemopreventive strategies.

Allelic heterogeneity in hereditary surfactant protein B (SP-B) deficiency.

Inability to produce surfactant protein B (SP-B) causes fatal neonatal respiratory disease. A frame-shift mutation (121ins2) is the predominant but not exclusive cause of disease. To determine the range of mechanisms responsible for SP-B deficiency, both alleles from 32 affected infants were characterized. Sixteen infants were homozygous for the 121ins2 mutation, 10 infants were heterozygous for the 121ins2 and another mutation, and six infants were homozygous for other mutations. Thirteen novel SP-B gene mutations were identified, which were not found in a control population. One novel mutation was found in two unrelated families. Surfactant protein expression was evaluated by immunohistochemistry and/or protein blotting. Absence of proSP-B and mature SP-B was associated with nonsense and frame-shift mutations. In contrast, proSP-B expression was associated with missense mutations, or mutations causing in-frame deletions or insertions, and low levels of mature SP-B expression were associated with four mutations. Extracellular staining for proSP-C and/or aberrantly processed SP-C was observed in lungs of all infants with SP-B gene mutations. Hereditary SP-B deficiency is caused by a variety of distinct mutations in the SP-B gene and may be associated with reduced, as well as absent, levels of mature SP-B, likely caused by impaired processing of proSP-B.

Olive-oil consumption and health: the possible role of antioxidants.

In the Mediterranean basin, olive oil, along with fruits, vegetables, and fish, is an important constituent of the diet, and is considered a major factor in preserving a healthy and relatively disease-free population. Epidemiological data show that the Mediterranean diet has significant protective effects against cancer and coronary heart disease. We present evidence that it is the unique profile of the phenolic fraction, along with high intakes of squalene and the monounsaturated fatty acid, oleic acid, which confer its health-promoting properties. The major phenolic compounds identified and quantified in olive oil belong to three different classes: simple phenols (hydroxytyrosol, tyrosol); secoiridoids (oleuropein, the aglycone of ligstroside, and their respective decarboxylated dialdehyde derivatives); and the lignans [(+)-1-acetoxypinoresinol and pinoresinol]. All three classes have potent antioxidant properties. High consumption of extra-virgin olive oils, which are particularly rich in these phenolic antioxidants (as well as squalene and oleic acid), should afford considerable protection against cancer (colon, breast, skin), coronary heart disease, and ageing by inhibiting oxidative stress.

Altered surfactant protein B levels in transgenic mice do not affect clearance of bacteria from the lungs.

To determine the role of surfactant protein B (SP-B) in bacterial clearance from the airways, three groups of mice expressing different levels of SP-B were studied: wild-type mice, hemizygous SP-B mice, and SP-B overexpressing transgenic mice. SP-B levels in overexpressing mice were increased 5-fold relative to hemizygous mice and 2- to 3-fold over wild-type littermates. Mice from each group were infected intratracheally with the common airway pathogens, group B streptococci or Pseudomonas aeruginosa. There was no significant difference in the number of recoverable viable bacteria at 6 h (group B streptococci and P. aeruginosa) and at 24 h (P. aeruginosa) among the three groups. Similarly, systemic dissemination of bacteria was not different among the three groups for both pathogens and at both time points. We conclude that SP-B levels in vivo do not influence clearance of bacteria from the lungs.

Serial changes in surfactant-associated proteins in lung and serum before and after onset of ARDS.

The goal of this study was to determine the changes that occur in surfactant-associated proteins in bronchoalveolar lavage fluid (BAL) and serum of patients at risk for ARDS and during the course of ARDS. We found that the concentrations of SP-A and SP-B were low in the BAL of patients at risk for ARDS before the onset of clinically defined lung injury, whereas the concentration of SP-D was normal. In patients with established ARDS, BAL SP-A and SP-B concentrations were low during the entire 14-d observation period, but the median SP-D concentrations remained in the normal range. Immunoreactive SP-A and SP-D were not increased in the serum of patients at risk for ARDS, but both increased after the onset of ARDS to a maximum on Day 3 and remained elevated for as long as 14 d. The BAL SP-A concentrations were significantly lower in at-risk patients who developed ARDS, and no patient with a BAL SP-A concentration greater than 1.2 microg/ml developed ARDS. On Days 1 and 3 of ARDS, the BAL SP-D concentration was significantly lower in patients who died, and the BAL SP-D concentration was significantly related to the PI(O(2))/FI(O(2)) ratio. Thus, surfactant protein abnormalities occur before and after the onset of ARDS, and the responses of SP-A, SP-B, and SP-D differ in important ways. The BAL SP-A and SP-D measurements can be used to classify patients as high or low risk for progression to ARDS and/or death after the onset of ARDS. Strategies to increase these surfactant proteins in the lungs of patients with ARDS could be useful to modify the onset or the course of ARDS.