Identification of Colchicine in Placental Blood from Patients Using

Hundreds of products described as “natural” and “harmless” are now available, which are supposed to treat a variety of human afflictions. As h...
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Identification of Colchicine in Placental Blood from Patients Using Herbal Medicines Howard R. Petty,* Menik Fernando, Andrei L. Kindzelskii, Bohdan N. Zarewych,‡ Mohamad B. Ksebati, Lew M. Hryhorczuk, and Shahriar Mobashery* Departments of Chemistry and Biological Sciences and the Institute for Drug Design, Wayne State University, Detroit, Michigan 48202, and Department of Gynecology and Obstetrics, Rochester Hospital, Rochester, Michigan 48307 Received April 11, 2001

While characterizing natural antiinflammatory substances in human placental blood, we discovered a factor that affected human neutrophils and their adherence. Rigorous chemical and stereochemical analyses revealed this factor to be the well-known alkaloid, colchicine. When samples from individual patients were analyzed, significant levels (49-763 µg/L) of colchicine could be found in placental blood of patients using nonprescription herbal dietary supplements during pregnancy. We confirmed the presence of colchicine in commercially available ginkgo biloba. Due to its potential harmful effects, it would appear that such supplements should be avoided by women who are pregnant or are trying to conceive.

Introduction The use of herbal medicines has risen dramatically recently in North America and Western Europe. In the United States, roughly a third of the adult population uses herbal medicines as food supplements, thus leading to a five billion dollar per year market. Hundreds of products described as “natural” and “harmless” are now available, which are supposed to treat a variety of human afflictions. As herbal usage has increased, so have the reported side effects. Hepatotoxicity is a common side effect of several herbs (1, 2). Midwives often recommend herbal treatments to alleviate the symptoms of pregnancy and induce labor (3). Unfortunately, the use of Caulophyllum thalictroides and Tripterygium wilfordii have been associated with congestive heart failure and occipital meningoencephalocele in neonates, respectively (4, 5). In general, the safety and efficacy of herbal medicines have not been verified by appropriate randomized and controlled clinical studies (6). Although various in vivo and in vitro side effects of herbal products have been noted, the chemical mechanisms involved are often unknown. During an analysis of neonatal neutrophil function, we discovered the well-known plant alkaloid, colchicine ((-)-N-5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy9-oxobenzo[a]heptalene-7-yl(S)-acetamide) (7, 8), in human placental blood. Although colchicine is a well-known product of the autumn crocus (Colchicum autumnale), it is also found in approximately 200 species of plants across 20 genera (7-9). Our discovery was entirely unanticipated because colchicine is used to block microtubule assembly in vitro (10). It also possesses antiinflammatory, anti-metastatic, and teratogenic activities * To whom correspondence may be addressed. (S.M.) Phone: (313) 577-3924. Fax: (313) 577-8822. E-mail: [email protected]. (H.R.P.) Phone: (313) 577-2896. Fax: (313) 577-6891. E-mail: hpetty@ biology.biosci.wayne.edu. ‡ Department of Gynecology and Obstetrics.

in vivo and can be fatal at high doses (7, 8, 11). In light of these activities, the discovery of colchicine in placental blood is cause for concern and merits further investigation.

Materials and Methods Authentic colchicine was purchased from the Aldrich Chemical Co. Acetonitrile was HPLC grade (Fisher). Water was deionized and distilled. Solvent mixtures were degassed before being used for HPLC. Mobile phases were prepared by filtering solvents through 0.45 µm nylon membrane filters (Gelman). HPLC columns (protein C4, particle size ) 5 µm, 1.0 × 25 cm; protein C4, particle size ) 5 µm, 0.46 × 25 cm; protein and peptide C18, particle size ) 5 µm, 0.46 × 25 cm) were purchased from Vydac. 1H NMR, 13C NMR, DEPT, HMQC, and NOE experiments were performed on a Varian 500 MHz spectrometer. FAB, EI, and CI mass spectra were recorded on a Kratos MS 80RFA spectrometer. IR spectra were obtained on a Nicolet DX instrument and circular dichroic spectra were recorded using a Jasco Model J600 spectrometer. FMLP-Mediated Assay. Neutrophils were separated from fresh peripheral blood from healthy volunteers using an IRBapproved protocol. They were then washed with Hank’s balance salt solution (HBSS). N-Formylmethionylleucylphenylalanine (FMLP) was dissolved initially in 20 µL of DMSO and was then diluted in HBSS to give a stock solution of 10-5 M. The samples from HPLC were evaporated to dryness and the residues were taken up in HBSS. Small amounts of purified HPLC fractions from placental blood were mixed with neutrophils. FMLP was added to these mixtures, then the solutions were incubated for 20-25 min at 37 °C. An aliquot of the FMLP stock solution was added to the neutrophils to make the final FMLP concentration 10-7-10-8 M. The metabolic flux, as judged by NAD(P)H oscillations, was observed using a microscope with a high sensitivity detector (12, 13). Neutrophils show 10-s NAD(P)H oscillations with FMLP, which decrease to 20-s with active compounds isolated from placental blood. The solution containing the active compound was diluted until the NAD(P)H oscillations changed to 10 s. Sample Preparation from Bulk Blood. Fresh blood was collected from the human umbilical cord and placenta after child

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Figure 1. (A) 1H NMR spectrum of the unknown compound (CDCl3, 500 MHz, room temperature). The peak at δ 7.27 corresponds to CHCl3. Resonances between δ 1.80-0.55 are small impurities in the sample. (B) 13C NMR spectrum of the unknown compound (D2O, 500 MHz, 10 °C) delivery under IRB approval. No special selection criteria were employed for inclusion in the study. The blood sample was centrifuged (400 g, 20 min) to remove all cells from the serum. Higher molecular weight proteins were removed by filtering the serum through an Amicon (3000) filter. The pH of the serum was adjusted to 8.0-8.3 by adding bicarbonate/carbonate buffer (0.1 M, pH 9.6) before concentrating the solution in vacuo at 45 °C. The sample size of 200 mL was concentrated to ∼1 mL for each injection. The concentrated sample was centrifuged (3000 rpm for 5 min) to remove salts and precipitated proteins. The clear solution was filtered through a 0.02-µm syringe filter just before injection into the HPLC. Sample Preparation from Blood from Individual Pregnancies. These experiments were performed after the identity of the natural product from the bulk placental blood was identified to be colchicine. The blood sample from each placenta measured ∼100-150 mL. Red blood cells and proteins were removed by centrifugation and filtration, as described above. To 100 mL of filtered serum was added 1 mL of 1 M (NH4)2HPO4 (pH 8), and colchicine was extracted into methylene chloride (100 mL, 3×) according to the procedure of Tracqui et

al. (15). The organic layer was dried over anhydrous MgSO4, the suspension was filtered, and the filtrate was concentrated to dryness in vacuo. The residue was dissolved in the mobile phase and was injected into the HPLC column. All samples were protected from light. HPLC System for Analysis of the Sample from Bulk Blood. A Perkin-Elmer 410 LC Bio pump attached to an LC95 variable-wavelength UV-vis detector and computerized Dynamax Macintegrater (Rainin Instrument Co., Inc.) were used. The detector was set at 230 nm and the flow rate was 1 mL/min. The first separation was carried out by a C4 reversedphase column (1.0 × 25 cm) with a linear gradient starting from 5% CH3CN in water (solvent A) to 95% CH3CN in water (solvent B). The column was initially washed for 10 min with solvent A after the injection of the sample into the column. Then, a linear gradient was established for transition from solvent A to B over 60 min. The column was subsequently washed for another 10 min with solvent B. The samples of purified compound from different runs were combined (tR ) 71 min) and further purified with an isocratic elution profile using the C4 reversed-phase column (0.46 × 25 cm) with 30% CH3CN in water (tR ) 46 min).

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HPLC System for the Quantitative Analysis of the Samples from Individual Pregnancies. The extracted colchicine from the blood sample was quantified on a C18 reversedphase (0.46 × 25 cm) column with an isocratic elution (mobile phase: 80% water, 15% CH3CN, 5% MeOH). Detection was made at 254 nm and the flow rate was 1 mL/min. Five different concentrations of authentic colchicine in the mobile phase (0.25, 0.5, 1.0, 1.5, and 2 mg/mL) were prepared and 10-µL portions were injected into the C18 reversed-phase HPLC. The standard curve was made by plotting concentrations versus the integrated areas under the colchicine peaks. Characterization of Colchicine from Blood Samples. 1H NMR (CDCl3, 500 MHz): δ 1.86, 2.28, 2.41, 2.54, 4.66 (all m, 5H); one CH3, δ 2.00 (s, 3H); four OCH3, δ 3.66, 3.91,3.95, 4.01 (all s, 12H); one NH, δ 6.66; δ 6.54 (s, 1H), δ 7.33 (d, J ) 11 Hz, 1H), δ 7.49 (s, 1H), δ 8.86 (d, J ) 11 Hz, 1H). 1H NMR (D2O, 500 MHz): δ 1.77, 1.97, 2.07, 2.40, 4.23 (all m, 5H); one CH3, 1.87 (s, 3H); four OCH3, δ 3.42, 3.74, 3.76, 3.79 (all s, 12H); δ 6.64 (s, 1H), δ 7.07 (d, J ) 11 Hz, 1H), δ 7.27 (d, J ) 11 Hz, 1H), δ 7.28 (s, 1H). 13C NMR and DEPT (D2O, 500 MHz): δ 22.1 (COCH3), 29.3 (CH2), δ 35.7 (CH2), δ 53.4 (CH-N), δ 56.4, 56.7, 61.8, 62.0, (4 OCH3), δ 108.5, 115.9, 125.1, 130.1 (4 CH), δ 135.9, 137.7, 138.0, 140.3, 150.2, 153.5, 154.0, 164.7 (8 C), δ 174.4 (NHCOCH3), 180.2 (CdO). IR (ZnSe, film) cm-1: 3345(m), 3060(w), 2960(m), 1630(s), 1580(s). UV (CH2Cl2): λmax 245 nm (4.38), 347 nm (4.04) HR-EI-MS: calcd for C22H25O6N, 399.1681; found, 399.1682; calcd for M - (CO+NHCOCH3+H+CH3), 297.1127; found, 297.1122; calcd for M - (CO+NHCOCH3+OCH3+H), 281.1721; found, 281.1719. LR-CI-MS: m/z (rel intensity) 400 (100, M + H), 85 (39), 61 (65); LR-FAB-MS: m/z (rel intensity) 400 (100, M + H), 422 (9, M + Na). CD (H2O) molar ellipticity: +684 (240 nm), -1710 (265 nm), -684 (320 nm), -1254 (350 nm), -228 (380 nm).

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The proton multiple quantum coherence spectrum (HMQC; Figure 2) demonstrated that these protons were attached to carbons as follows: the proton at δ 4.66 was attached to the carbon at δ 53.39, two protons (δ 1.81 and 2.26) were attached to the carbon with δ 35.68, and the last two protons (δ 2.43, 2.56) were attached to the carbon with δ 29.25. From this information, the presence of a methine and two methylenes was discerned. The carbon-to-carbon connectivity was confirmed by a homodecoupling experiment and it established the following partial structure (-CH-CH2-CH2-). The most deshielded carbon (δ 53.39) in this partial structure should be attached to the acetamide group. The mass spectrum showed a mass fragment that could correspond to [M CO]+, which suggested a tropolone ring system. The 1H NMR spectrum (Figure 1) clearly showed four different methoxyl groups (δ 3.66, 3.91, 3.95, and 4.00). The chemical shifts of three of the four methoxyls are very close and one (δ 3.66) is different. We concluded that the three methoxyls may be on one ring and the fourth should be on another ring. The DEPT spectra showed five methyl, five methine, and two methylene groups, and the remainder were quaternary carbons. These data collectively provided information for two possible structures, 1 or 2.

Results and Discussion Approximately 3 L of human placental blood (from 30 pregnancies) was collected after childbirth from two hospitals on each of four different occasions. The cellfree serum was processed by ultrafiltration using a molecular weight cutoff of 3 kDa. The filtered samples were purified by HPLC, while assaying the ability of fractions to block N-formylmethionyl-leucyl-phenylalanine (FMLP)-mediated activation of metabolic flux (12, 13). This assay is based on the cytosolic NAD(P)H autofluorescence oscillation of human neutrophils. A paleyellow compound (700 µg) was isolated. The empirical formula (C22H25O6N) for this compound was established by high-resolution EI mass spectrometry (experimental 399.1682, calculated 399.1681). This determination was followed by NMR experiments (Figure 1), which showed 22 different carbons and 25 different hydrogens. The degree of unsaturation for this compound was calculated from the molecular formula to be 11. The IR spectrum showed two absorption peaks near 1600 cm-1, suggesting that these could be attributed to two conjugated carbonyl groups in the unknown compound. This was confirmed by 13C and DEPT NMR data [conjugated carbonyl δ 180 (s); acetamide δ 174.4 (s); 1H NMR (in CDCl3): δ 6.66 (s, 1 H, exchanged with D2O), δ 2.0 (s, CH3)]. The 13C NMR spectrum provided evidence for six carbon-to-carbon double bonds (δ 164.9, 154.0, 153.7, 150.4, 140.6, 138.1, 137.8, 136.1, 130.2, 125.4, 116.1, 108.7). These functionalities account for eight of the eleven degrees of unsaturation. The remaining three degrees of unsaturation are accounted for by three ring systems (two aromatic and one alicyclic). The 1H NMR spectrum (D2O) showed five different aliphatic multiplets at δ 1.81, 2.26, 2.43, 2.56, and 4.66.

NOE experiments were performed to confirm the proton connectivities. The measured NOEs were consistent with structure 1 and are given for the more detailed structure 1a. A search of the literature revealed 1 to be

colchicine. The stereochemistry of the carbon bearing the acetamide group was determined by circular dichroic measurements and comparison with an authentic colchicine sample. The stereogenicity of this carbon was found

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Figure 2. Proton multiple quantum coherence (HMQC) spectrum of the unknown compound (D2O, 500 MHz, 10 °C).

to be S. The identity of the compound was further confirmed by demonstration of the identity for the NMR, UV, IR, and MS (EI, CI, FAB) spectra of an authentic sample of colchicine to the data obtained from the isolated compound. During our NMR structural analysis of the unknown compound, we found that the proton chemical shifts depended upon both concentration and temperature. This unusual feature led us to believe that the compound was degrading in the course of purification. But apparently, these are features seen with colchicine, as reported in the literature (14) and also confirmed by us with the authentic sample. This unusual behavior appears to be explained by the subtle conformational differences that colchicine can assume (14). Having identified cholchicine in the placental blood, we looked at literature reports for its isolation and quantification. A report of colchicine extraction into methylene chloride has appeared in the literature (15). This method worked well in our hands, eliminating the need for HPLC purification. Having developed the HPLC assay for the purification step, this technique served as an effective method for quantification of the amount of colchicine in each sample. For these individual analyses, we used 24 different blood samples from 24 pregnancies. The blood from each sample was extracted with methylene chloride; then the organic extract was analyzed for

its colchicine content. The amount of colchicine was analyzed against a standard plot for colchicine of known concentrations. Our experimental results clearly showed that 5 of the 24 samples contained colchicine (760, 182, 106, 97, and 49 µg/L). All of the colchicine-containing (>2 µg/L) blood samples came from women who used herbal supplements. Women who described themselves as nonusers of herbal supplements (both vegetarians and nonvegetarians) had little or no detectable colchicine in their placental blood samples. Thus, the use of herbal supplements correlates with the appearance of colchicine in placental blood. Since herbal medicine usage was correlated with the appearance of colchicine in placental blood, we next sought to directly test for the presence of colchicine in these preparations. We purchased the commercial ginkgo biloba and echinacea in the Detroit area. Samples were extracted and analyzed as described above. Significant levels of colchicine (26 ( 3 µg/tablet) were found for the commercially available ginkgo biloba sample. However, echinacea tablets had little colchicine (2.0 ( 0.5 µg/ tablet). The identity of colchicine in the ginko biloba extract was re-confirmed by mass spectroscopy (data not shown). Therefore, the appearance of colchicine in the placental blood of herbal medicine users in the Detroit area is consistent with its presence in ginkgo biloba tablets.

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The accumulation of colchicine in placental serum could not be accounted for by prescriptional use, as the sample donors were not undergoing the rare clinical treatments that use colchicine. Since mammals do not synthesize colchicine, the observed accumulation of colchicine is likely due to ingestion of plant materials. However, significant levels of colchicine were only found in the placental blood of herbal medicine users, and no other individuals, including vegetarians. The presence of colchicine was confirmed in ginkgo biloba. We speculate that, over the course of a pregnancy, colchicine from herbal supplements accumulates in placental blood. The rapidly growing fetus may be particularly vulnerable to the antimitotic effects of colchicine. Hence, fetuses carried by mothers ingesting high levels of herbal medications are potentially at risk. Biological effects in vitro can be observed in the range of 1-100 µM (0.4-40 mg/L), (16) which includes the range observed in placental blood (e0.76 mg/L). Recent studies have suggested that certain herbal substances may have a detrimental effect on reproduction and fetal health, and may cause birth defects (1-5, 17, 18). The presence of significant levels of colchicine in placental blood may contribute to these observations. The potential detrimental roles of natural plant products in reproductive health merit further analysis.

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Meningoencephalocele associated with Tripterygium wilfordii treatment. Pediatr. Neurosurg. 27, 45-48. Vogler, B. K., Pittler, M. H., and Ernst, E. (1999) The efficacy of ginseng. A systematic review of randomised clinical trials. Eur. J. Clin. Pharmacol. 55, 567-575. Wildman, W. C. (1960) Colchicine and related compounds. In The Alkaloids (Manske, R. H. F., Ed.) Vol. 6, pp 247-278, Academic Press, NY. Wildman, W. C., and Pursey, B. A. (1968) Colchicine and related compounds. In The Alkaloids (Manske, R. H. F., Ed.) XI, pp 407457, Academic Press, NY. Dahlgren, R. (1985) Liljor och liljetra¨d. Svensk. Bot. Tidskr. 79, 1-16. Petty, H. R. (1993) Molecular Biology of Membranes, Plenum Press, NY. Simons, R., and Kingma, D. W. (1989) Fatal colchicine toxicity. Am. J. Med. 86, 356-357. Kindzelskii, A. L., Eszes, M. M., Todd, R. F., III., and Petty, H. R. (1997) Proximity oscillations of complement receptor type 4 and urokinase receptors on migrating neutrophils are linked with signal transduction/metabolic oscillations. Biophys. J. 73, 17771784. Kindzelskii, A. L., Zhou, M. J., Haugland, R. P., Boxer, L. A., and Petty, H. R. (1998) Oscillatory pericellular proteolysis and oxidant deposition during neutrophil migration. Biophys. J. 74, 9097. Berg, U., Bladh, H., Hoff, M., and Svensson, C. (1997) Stereochemical variations on the colchicine motif. Part 2. Unexpected tetracyclic isoxazole derivatives. J. Chem. Soc., Perkin Trans. 2, 1697-1704. Tracqui, A., Kintz, P., Ludes, B., Rouge, C., Douibi, H., and Mangin, P. (1996) High-performance liquid chromatography coupled to ion spray mass spectrometry for the determination of colchicine at ppb levels in human biofluids. J. Chromatogr. B 675, 235-242. Schellenberg, R., and Gillespie, E. (1977) Colchicine inhibits phosphatidylinositol turnover induced in lymphocytes by concanavalin A. Nature 265, 741-742. Ondrizek, R. R., Chan, P. J., Patton, W. C., and King, A. (1999) Inhibition of human sperm motility by specific herbs used in alternative medicine. J. Assist. Reprod. Genet. 16, 87-91. Ondrizek, R. R., Chan, P. J., Patton, W. C., and King, A. (1999) An alternative medicine study of herbal effects on the penetration of zona-free hamster oocytes and the integrity of sperm deoxyribonucleic acid. Fertil. Steril. 71, 517-522.

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