Bioactive Properties of the Aqueous Extracts of Endophytic Fungi Associated with Scots Pine (Pinus sylvestris) Roots.

Despite the continuing interest in various plant and natural products, only a small portion of the biologically active compounds from nature has been discovered and exploited. In this study, antioxidant and antibacterial properties of aqueous fractions of three endophytic fungi isolated from the roots of 8-year-old Scots pines (Pinus sylvestris) growing on a drained peatland were investigated. The endophytic fungi species were Acephala applanata, Phialocephala fortinii, and Humicolopsis cephalosporioides/Coniochaeta mutabilis. The bioactivities were examined using hydrogen peroxide scavenging and oxygen radical absorbance capacity tests as well as sensitive Escherichia coli-based biosensors, which produce a luminescent signal in the presence of substances with oxidative or genotoxic properties. In addition, cell models for Parkinson's disease, age-related macular degeneration, and osteoarthritis were used to evaluate the potential for pharmaceutical applications. The aqueous extracts of fungi and 19 out of 42 fractions were found to be active in one or more of the tests used. However, no activity was found in the age-related macular degeneration and osteoarthritis cell model tests. Additionally, bioactivity data was connected with metabolites putatively annotated, and out of 330 metabolites, 177 were interesting in view of the bioactivities investigated. A majority of these were peptides and all three fungal species shared a highly similar metabolome. We propose that Scots pine endophytic fungi are a rich source of interesting metabolites, and synergistic effects may cause the bioactivities, as they were found to vary after the fractionation process.

Consumption of chokeberry (Aronia mitschurinii) products modestly lowered blood pressure and reduced low-grade inflammation in patients with mildly elevated blood pressure.

Previous studies suggest that consumption of chokeberries may improve cardiovascular disease risk factor profiles. We hypothesized that chokeberries (Aronia mitschurinii) have beneficial effects on blood pressure, low-grade inflammation, serum lipids, serum glucose, and platelet aggregation in patients with untreated mild hypertension. A total of 38 participants were enrolled into a 16-week single blinded crossover trial. The participants were randomized to use cold-pressed 100% chokeberry juice (300 mL/d) and oven-dried chokeberry powder (3 g/d), or matched placebo products in random order for 8 weeks each with no washout period. The daily portion of chokeberry products was prepared from approximately 336 g of fresh chokeberries. Urinary excretion of various polyphenols and their metabolites increased during the chokeberry period, indicating good compliance. Chokeberries decreased daytime blood pressure and low-grade inflammation. The daytime ambulatory diastolic blood pressure decreased (-1.64 mm Hg, P = .02), and the true awake ambulatory systolic (-2.71 mm Hg, P = .077) and diastolic (-1.62 mm Hg, P = .057) blood pressure tended to decrease. The concentrations of interleukin (IL) 10 and tumor necrosis factor α decreased (-1.9 pg/mL [P = .008] and -0.67 pg/mL [P = .007], respectively) and tended to decrease for IL-4 and IL-5 (-4.5 pg/mL [P = .084] and -0.06 pg/mL [P = .059], respectively). No changes in serum lipids, lipoproteins, glucose, and in vitro platelet aggregation were noted with the chokeberry intervention. These findings suggest that inclusion of chokeberry products in the diet of participants with mildly elevated blood pressure has minor beneficial effects on cardiovascular health.

Rapid chemical characterisation of stilbenes in the root bark of Norway spruce by off-line HPLC/DAD-NMR.

INTRODUCTION: Stilbenes are plant secondary metabolites that have shown promising and varied biological activities. Stilbenes are presently actively studied for the exploitation of this primary raw material resource, involving the concept of biorefining. Methods for the rapid discovery of new and known stilbene structures from various plant sources are thus keenly sought. OBJECTIVE: To establish a simple and rapid technique of off-line HPLC with a diode-array detector (DAD) and NMR for the unambiguous structural elucidation of stilbene structures in the root bark of Norway spruce [Picea abies (L.) Karst.]. MATERIAL AND METHODS: The stilbene containing fraction was extracted from the plant bark with an ethanol:water mixture (95:5, v/v) preceded by defatting of hydrophobic compounds with n-hexane using the accelerated solvent extraction technique. A portion of the ethanol-water soluble extract was hydrolysed with ß-glucosidase to prepare stilbene aglycones. The extracts were further purified and enriched using a polymeric adsorbent. Stilbene-enriched extracts were directly characterised by off-line HPLC/DAD-NMR in conjunction with HPLC/DAD and HPLC/DAD with electrospray ionisation MS(n). RESULTS: Trans-isorhapontin and trans-astringin were identified as the major, and trans-piceid as a minor, stilbene glucosides of the bark of roots of Picea abies. Not only stilbene glucosides but also the corresponding stilbene aglycones, such as trans-resveratrol, trans-piceatannol and trans-isorhapontigenin, were rapidly identified from the hydrolysed extract. The acquired heteronuclear single-quantum coherence and heteronuclear multiple bond correlation spectra were used to assign the complete carbon NMR chemical shifts of trans-isorhapontin and trans-astringin without the need of acquiring a (13)C-NMR spectrum. CONCLUSION: The off-line HPLC/DAD-NMR method is expedient for the unambiguous identication of structurally similar stilbenes in plant extracts.

Transcriptional activity of estrogen-related receptor γ (ERRγ) is stimulated by the phytoestrogen equol.

Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor lacking identified natural ligands. The synthetic estrogen receptor ligands 4-hydroxytamoxifen and diethylstilbestrol have, however, been shown to bind to and abolish the constitutive transcriptional activity of ERRγ. Certain phytoestrogens were recently reported to act as agonists of the related ERRα. We investigated whether phytoestrogens also modulated the transcriptional activity of ERRγ. We analyzed a selection of phytoestrogens for their potential agonistic or antagonistic activity on ERRγ. In transiently transfected PC-3 and U2-OS cells equol stimulated the transcriptional activity of ERRγ and enhanced its interaction with the coactivator GRIP1. The agonistic effect of equol was abolished by 4-hydroxytamoxifen. Equol induced a conformational change in the ERRγ ligand-binding domain. Based on structural models of the ERRγ ligand-binding domain, we were able to introduce mutations that modulated the agonistic potential of equol. Finally, equol enhanced the growth inhibitory effect of ERRγ on the prostate cancer PC-3 cells. In conclusion, we have demonstrated that the phytoestrogen equol acts as an ERRγ agonist.

Equol in milk of dairy cows is derived from forage legumes such as red clover.

The intake of isoflavones and the resulting equol contents of both plasma and milk of the same red clover-fed cows are reported for the first time in cyclic change-over design study. Cows were fed four different red clover silages and two timothy-meadow fescue silages as controls. The red clover silages contained daidzein, formononetin, biochanin A and genistein, whereas the timothy-meadow fescue silages contained no isoflavones. We found a strong association (y = 0.071x+2.75, R 2 0.71) between the formononetin intake (x) and equol concentration (y) in the plasma, while the formononetin intake and milk equol concentration were weakly associated (y = 0.0035x+0.358, R 2 0.20). This means that a small part of the total formononetin in the silage is secreted into milk as equol. The mean equol contents in plasma and milk of cows fed red clover silage diets were in the range of 4.6-8.4 mg/l and 458-643 microg/l, respectively, while the respective values for the control diets were in the range of 0.8-1.5 mg/l and 171-287 microg/l. We showed that shorter growing periods of red clover resulted in higher silage formononetin contents and plasma and milk equol contents, suggesting that the equol content of milk can be manipulated by varying the harvesting strategy of red clover. We conclude that milk equol is derived from the formononetin of red clover silage and that milk from red clover-fed cows can be considered as a source of equol in human nutrition.

The binding of lignans, flavonoids and coumestrol to CYP450 aromatase: a molecular modelling study.

Androgens are transformed into aromatic estrogens by CYP450 aromatase in a three-step reaction consuming three equivalents of oxygen and three equivalents of NADPH. Estrogens are substrates for nuclear estrogen receptors (ERs) and play a key role in estrogen-dependent tumour cell formation and proliferation. Natural phytoestrogens are proved to be competitive inhibitors of aromatase enzyme at IC(50) values in micromolar levels. In order to understand the mechanisms involved in the binding of various phytoestrogens, we used our model of CYP450 aromatase to study the binding of phytoestrogens using molecular dynamics simulations with a bound phytoestrogen. The simulation trajectory was analysed to find the essential interactions which take place upon binding and a representative structure of the trajectory was minimized for docking studies. Sets of phytoestrogens, such as lignans, flavonoids/isoflavonoids and coumestrol, were docked into the aromatase active site and the binding modes were studied.

Food frequency questionnaires and overnight urines are valid indicators of daidzein and genistein intake in U.S. women relative to multiple 24-h urine samples.

Data regarding convenient, valid methods for measuring U.S. isoflavone intake are limited. We evaluated a soy food questionnaire (SFQ), the Willett food frequency questionnaire (FFQ), and overnight urine samples relative to excretion in 24-h urine samples. We also described intake among women in a high-risk program for breast or ovarian cancer. Between April 2002 and June 2003, 451 women aged 30 to 50 yr with a family history of breast or ovarian cancer completed the SFQ and FFQ. Of them, 27 provided four 24-h and overnight urine specimens. In these women, 24-h sample measures were correlated with SFQ estimates of daidzein (Spearman r = .48) and genistein (r = .54) intake, moderately correlated with the Willett FFQ (daidzein r = .38, genistein r = .33), and strongly correlated with overnight urine excretion (daidzein r = .84, genistein r = 0.93). Among all 451 SFQ respondents, mean (median) daidzein and genistein intakes were 2.8 (0.24) and 3.9 (0.30) mg/day. Primary sources of both were soymilk, soy nuts, and tofu. We conclude that targeted soy food questionnaires, comprehensive FFQs, and multiple overnight urines are all reasonable options for assessing isoflavone intake in epidemiologic studies.

Long-term consumption of isoflavone-enriched foods does not affect bone mineral density, bone metabolism, or hormonal status in early postmenopausal women: a randomized, double-blind, placebo controlled study.

BACKGROUND: Osteoporosis is a major health problem. It was hypothesized that isoflavone-containing products may be a potential alternative to hormone replacement therapy for preventing bone loss during the menopausal transition. OBJECTIVE: The objective was to investigate whether the consumption of isoflavone-enriched foods for 1 y affects bone mineral density, bone metabolism, and hormonal status in early postmenopausal women. DESIGN: This was a randomized, double-blind, placebo-controlled, parallel, multicenter trial. Two hundred thirty-seven healthy early postmenopausal women [mean (+/-SD) age of 53 +/- 3 y and time since last menses of 33 +/- 15 mo] consumed isoflavone-enriched foods providing a mean daily intake of 110 mg isoflavone aglycones or control products for 1 y while continuing their habitual diet and lifestyle. Outcome measures included bone mineral density of the lumbar spine and total body, markers of bone formation and bone resorption, hormones, isoflavones in plasma and urine, safety variables, and adverse events. RESULTS: Consumption of isoflavone-enriched products did not alter bone mineral density of the lumbar spine and total body or markers of bone formation and bone resorption. Hormone concentrations did not differ between the isoflavone and control groups. Consumption of isoflavone-enriched products resulted in increased isoflavone concentrations in plasma and urine, whereas control products did not. This finding indicated good compliance with treatment. Subgroup analysis did not support an effect of equol phenotype on bone density. The intervention had no effect on a range of safety variables and reported adverse events. CONCLUSION: Consumption of foods containing 110 mg/d of soy isoflavone aglycone equivalents for 1 y did not prevent postmenopausal bone loss and did not affect bone turnover in apparently healthy early postmenopausal white women. This trial was registered at clinicaltrials.gov as NCT00301353.

Inhibitory effects of olive oil phenolics on invasion in human colon adenocarcinoma cells in vitro.

Studies in human, animal and cellular systems suggest that phenols from virgin olive oil are capable of inhibiting several stages in carcinogenesis, including metastasis. The invasion cascade comprises cell attachment to extracellular matrix components or basement membrane, degradation of basement membrane by proteolytic enzymes and migration of cells through the modified matrix. In the present study, we investigated the effect of phenolics extracted from virgin olive oil (OVP) and its main constituents: hydroxytyrosol (3,4-dihydroxyphenylethanol), tyrosol (p-hydroxyphenylethanol), pinoresinol and caffeic acid. The effects of these phenolics were tested on the invasion of HT115 human colon carcinoma cells in a Matrigel invasion assay. OVP and its compounds showed different dose-related anti-invasive effects. At 25 microg/ml OVP and equivalent doses of individual compounds, significant anti-invasive effects were seen in the range of 45-55% of control. Importantly, OVP, but not the isolated phenolics, significantly reduced total cell number in the Matrigel invasion assay. There were no significant effects shown on cell viability, indicating the reduction of cell number in the Matrigel invasion assay was not due to cytotoxicity. There were also no significant effects on cell attachment to plastic substrate, indicating the importance of extracellular matrix in modulating the anti-invasive effects of OVP. In conclusion, the results from this study indicate that phenols from virgin olive oil have the ability to inhibit invasion of colon cancer cells and the effects may be mediated at different levels of the invasion cascade.

Synthesis of daidzein 7-O-beta-D-glucuronide-4'-O-sulfate.

The first synthesis of daidzein 7-O-beta-D-glucuronide-4'-O-sulfate, a mixed conjugate of an important dietary phytoestrogen is described.

Lignan and isoflavone excretion in relation to uterine fibroids: a case-control study of young to middle-aged women in the United States.

BACKGROUND: Uterine fibroids are hormonally responsive; estradiol and progesterone stimulate their growth, and gonadotrophin-releasing hormone agonists shrink them. Phytoestrogens, including isoflavones and lignans, can act as weak estrogens or antiestrogens. OBJECTIVE: The objective of this case-control study was to evaluate the relation between uterine fibroid risk and phytoestrogen exposure. DESIGN: Two overnight urine collections (48 h apart) from 170 uterine fibroid cases and 173 controls were analyzed for isoflavonoids (ie, daidzein, genistein, equol, and O-desmethylangolensin) and lignans (enterodiol and enterolactone). Logistic regression was used to determine associations between the mean excretion of the 2 collections and the risk of uterine fibroids. RESULTS: Unadjusted isoflavone excretion did not differ significantly between cases and controls (2.33 +/- 5.82 and 2.60 +/- 5.90 nmol/mg Cr, respectively; P = 0.68), but cases excreted significantly less lignans than did controls (2.86 +/- 3.45 and 4.57 +/- 6.67 nmol/mg Cr, respectively; P < 0.01). The trend for a reduced risk of uterine fibroids with increasing quartiles of lignan excretion was significant (odds ratio for highest versus lowest quartile = 0.31; 95% CI: 0.17, 0.58; P for trend < 0.01). When adjusted for age, BMI, race, family history of uterine fibroids, and isoflavone excretion, this trend remained but was attenuated (P = 0.07). CONCLUSIONS: Our findings suggest a modest inverse association between lignan excretion and uterine fibroid risk. Whether this relation represents an effect of lignans per se or of other constituents of lignan-containing foods on the development of uterine fibroids remains to be determined. No association was found between isoflavone excretion and uterine fibroids; however, the intake of soy foods, the primary source of isoflavones, was low in this population.

Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro.

It has been postulated that the R- and S-equol enantiomers have different biological properties given their different binding affinities for the estrogen receptor. S-(-)equol is produced via the bacterial conversion of the soy isoflavone daidzein in the gut. We have compared the biological effects of purified S-equol to that of racemic (R and S) equol on breast and prostate cancer cells of varying receptor status in vitro. Both racemic and S-equol inhibited the growth of the breast cancer cell line MDA-MB-231 (> or = 10 microM) and the prostate cancer cell lines LNCaP (> or = 5 microM) and LAPC-4 (> or = 2.5 microM). The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30 microM) was unable to prevent DNA damage in MCF-7 or MCF-10A breast cells following exposure to 2-hydroxy-4-nonenal, menadione, or benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. In contrast, racemic equol (10, 30 microM) prevented DNA damage in MCF-10A cells following exposure to 2-hydroxy-4-nonenal or menadione. These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol.

Reduced isoflavone metabolites formed by the human gut microflora suppress growth but do not affect DNA integrity of human prostate cancer cells.

Dietary isoflavones, such as genistein and daidzein, are metabolised by the human gut microflora. Case-control studies have disclosed a link between the formation of the daidzein metabolite equol and prostate cancer risk. We evaluated the effects of genistein, daidzein and five metabolites on two prostate cancer cell lines by determining DNA integrity and cell growth. LNCaP cells contain the T877A androgen receptor mutation whereas Los Angeles prostate cancer (LAPC)-4 cells express the wild-type receptor, both of which may affect responses to isoflavones. DNA integrity was determined using the comet assay. Cell growth was assessed by staining DNA with 4',6'-diamidino-2-pheylindole hydrochloride. Endogenous steroid hormones, but not isoflavones, induced DNA strand breaks. Dihydrotestosterone stimulated the growth of both cell lines. 17beta-Oestradiol increased the growth of LNCaP but not LAPC-4 cells, pointing to an involvement of the T877A androgen receptor. Isoflavones did not stimulate growth in either prostate cancer cell line. However, the growth of LNCaP and LAPC-4 cells was suppressed by genistein (inhibitory concentration 50 % (IC50) 39.7 mumol/l, 37.2 mumol/l) and by equol (IC50 53.8 mumol/l, 35.1 mumol/l). O-desmethylangolensin inhibited the growth of LAPC-4 cells (IC50 45.2 mumol/l), but not of LNCaP cells. In conclusion, isoflavones do not damage DNA or promote growth of androgen-dependent prostate cancer cells. Several isoflavones, including the reduced daidzein metabolites equol and O-desmethylangolensin, suppress cancer cell growth. Taken together, these data suggest a contribution of gut-formed isoflavone metabolites to the beneficial effects of dietary isoflavones on prostate cancer risk.

High concordance of daidzein-metabolizing phenotypes in individuals measured 1 to 3 years apart.

Particular intestinal bacteria are capable of metabolizing the soya isoflavone daidzein to equol and/or O-desmethylangolensin (O-DMA), and the presence of these metabolites in urine after soya consumption are markers of particular intestinal bacteria profiles. Prevalences of equol producers and O-DMA producers are approximately 30-50 % and 80-90 %, respectively, and limited observations have suggested that these daidzein-metabolizing phenotypes are stable within individuals over time. Characterizing stability of these phenotypes is important to understand their potential as markers of long-term exposure to particular intestinal bacteria and their associations with disease risk. We evaluated concordance within an individual for the equol-producer and O-DMA-producer phenotypes measured at two time points (T1, T2), 1-3 years apart. Phenotypes were ascertained by analysing equol and O-DMA using GC-MS in a spot urine sample collected after 3 d soya (source of daidzein) supplementation. In ninety-two individuals without recent (within 3 months before phenotyping) or current antibiotics use, 41 % were equol producers at T1 and 45 % were equol producers at T2, and 90 % were O-DMA producers at T1 and 95 % were O-DMA producers at T2. The percentage agreement for the equol-producer phenotype was 82 and for the O-DMA-producer phenotype was 89. These results indicate that these phenotypes are stable in most individuals over time, suggesting that they provide a useful biomarker for evaluating disease risk associated with harbouring particular intestinal bacteria responsible for, or associated with, the metabolism of the soya isoflavone daidzein.

Identification of urinary metabolites of the red clover isoflavones formononetin and biochanin A in human subjects.

Dietary supplements manufactured from red clover are widely marketed to provide the beneficial health effects of isoflavones without changing the original diet. In this study the metabolism of formononetin and biochanin A, the principal isoflavones of red clover, was studied in human subjects. Seven women ingested four red clover dietary supplements, and the metabolites of the isoflavones were identified in their urine samples. The structures of trimethylsilyl derivatives of the metabolites were established by GC-MS. New reduced metabolites of formononetin (dihydroformononetin and angolensin) and biochanin A (dihydrobiochanin A and 6'-hydroxyangolensin) were identified in urine samples using authentic reference compounds. Possible metabolic pathways are presented for the red clover isoflavones formononetin and biochanin A.

Synthesis of phytoestrogenic isoflavonoid disulfates.

Di-O-sulfates of six phytoestrogenic isoflavonoids, daidzein (1), genistein (2), glycitein (3), and the reduced metabolites dihydrodaidzein (4), dihydrogenistein (5) and equol (6) were synthesized. These compounds are known or potential inhibitors of steroid sulfatase enzymes. The new compounds were characterized by NMR and mass spectrometry.

An isotope dilution gas chromatographic-mass spectrometric method for the simultaneous assay of estrogens and phytoestrogens in urine.

The metabolism of endogenous estrogens is complicated and certain metabolic patterns may reflect an individual risk of estrogen-dependent diseases such as breast cancer. Since the 1960s we have been constantly involved in developing estrogen profiling methods, in the beginning using gas chromatography and later gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode (SIM) and finally utilizing isotope dilution (ID-GC-MS-SIM). The addition of the dietary phytoestrogens to the profile rendered the method even more complicated. The present work presents the final estrogen profile method for 15 endogenous estrogens, four lignans, seven isoflavonoids and coumestrol in one small urine sample (1/150th of a 24 h human urine sample, minimum 2.5-5 ml) with complete validation including investigations as to the precision, sensitivity, accuracy and specificity. The method does not include the minimal amounts of unconjugated estrogens in urine. It may also be used for animal (e.g. rat and mouse) urine using a minimum of 2 ml of usually pooled sample. Despite its complexity it was found to fulfill the reliability criteria, resulting in highly specific and accurate results.

Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton.

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases. Soy and soy foods are rich sources of isoflavones, which have been shown to possess several biological activities. In this study, the metabolism of soy isoflavones daidzein, genistein and glycitein was investigated in human subjects. The aim was to find and identify urinary phase I metabolites of isoflavones, which have an intact isoflavonoid skeleton, and which might possess some bioactivity. Six volunteers included three soy bars per day into their normal western diet for a 2-week period. Daily urine samples were collected before, and after the supplementation period. Urine samples were hydrolyzed with Helix pomatia, extracted with diethyl ether, purified with Sephadex LH-20 chromatography, and analyzed as trimethylsilyl derivatives using gas chromatography-mass spectrometry (GC-MS). The structures of the isoflavone metabolites were identified using authentic reference compounds. The metabolites, for which authentic reference compounds were not available, were identified by the interpretation of mass spectra. Several new isoflavone metabolites were identified, and the presence of previously reported metabolites confirmed. The metabolic pathways of daidzein, genistein and glycitein are presented on the basis of the identification of the metabolites in human urine after soy supplementation.

Analysis of phyto-oestrogens in biological matrices.

A review covering different methods for the analysis of phyto-oestrogens in biological matrices is presented. Sample pretreatment and analysis of isoflavonoids and lignans by HPLC and GC with various detection methods are discussed. The immunoassay method is also briefly presented.

Phyto-oestrogen levels in foods: the design and construction of the VENUS database.

The objective of the Vegetal Estrogens in Nutrition and the Skeleton (VENUS) project was to evaluate existing data on dietary exposure to compounds with oestrogenic and anti-oestrogenic effects present in plant foods as constituents or contaminants, and to identify and disseminate in vitro and in vivo methodologies to analyse the effects of such compounds on bone. To permit the assessment of exposure to isoflavones in European populations (Italy, the UK, Ireland, The Netherlands), the VENUS database of phyto-oestrogen levels in foods was established. Data on the isoflavone (genistein and daidzein) content of 791 foods, including almost 300 foods commonly consumed in Europe, were collected. Levels of coumestrol, formononetin and biochanin A in a limited number of foods were also included. Lignan levels (secoisolariciresinol and matairesinol) in 158 foods were incorporated into the database, which also contains information on the references sourced for the compositional data, on the analytical methods used by each author and on the number of foods analysed in each reference. The VENUS database was constructed in Microsoft Access 2000, which is widely available as part of Microsoft Office Professional. This paper outlines the procedures used for the selection and evaluation of existing literature data for incorporation into the database. In addition, the design of the database is described, along with the data entry and quality control procedures used in its construction. Limitations of the data are discussed and guidelines for its use are provided.