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Woolley, D. W., and Shaw, E. N.: Antiserotonins in Hypertension and the Antimetabolite Approach to Chemotherapy. Science 124: 34 July 6 ; , 1956.

Titrating heparin dosage via plasma anti-Xa levels. Mice were injected subcutaneously with 100 AL unfractionated heparin, Tinzaparin, or Fondaparinux diluted in PBS at various final concentrations. Thirty minutes later, blood was collected by cardiac puncture into 1 mL syringe containing 30 AL of mmol L EDTA. Samples were centrifuged twice at 2, 000 relative centrifugal force at 24jC to collect the plasma, which was stored at 80jC until analysis for anti-Xa activity. Human Antithrombin III 3.3 Ag well; Enzyme Research Laboratories, South Bend, IN ; and human factor Xa 0.02 Ag well; Enzyme Research Laboratories ; , in 155 AL of 25 mmol L HEPES 150 mmol L NaCl pH 7.5 ; , were added to 1.25 AL plasma samples, which were then incubated with 25 Ag well of a synthetic factor Xa chromogenic substrate Diapharma, Columbus, OH ; . The reaction was stopped after 15 minutes by adding 50 AL well 20% acetic acid. The resulting chromophore was measured at 405 nm. Plasma heparin levels were calculated in anti-Xa units mL by comparing against a standard curve of heparin-spiked mouse plasma samples. Standards and samples were analyzed in triplicate. Final amounts used for 1 dosing were 6.56 units unfractionated heparin, 7.32 IU Tinzaparin, and 0.0033 mg Fondaparinux. The 3 dosing used thrice the amount. Carcinoma experimental metastasis assay. Mice were injected subcutaneously with 100 AL PBS or heparin in 100 AL PBS. Thirty minutes afterwards, 500, 000 MC38GFP cells were injected i.v. into the lateral tail vein. Mice from each studied group were injected in alternating order and cells were resuspended by gently flicking the tube before aspirating the sample for each injection. Twenty-seven days after injection, the mice were euthanized, the lungs were removed, and enhanced green fluorescent protein fluorescence in lysates was quantified as described 14 ; . Melanoma experimental metastasis assay. Mice were injected with heparin and 500, 000 B16F1 cells using the protocol described for the carcinoma metastasis assay. Seventeen days after injection, mice were euthanized, tracheal perfusion with 10% buffered formalin was done, and the lungs were removed and placed into 10% buffered formalin. Lungs were allowed to fix for a minimum of 24 hours, removed from formalin individually, and photographed using a digital camera. Lung weights were determined by removing the lungs from formalin, briefly setting them on filter paper to remove excess liquid, and then weighing them on a Sartorius analytic scale. Heparin disaccharide analysis. Disaccharide analysis was done by the University of California, San Diego, Glycotechnology Core Facility. Briefly, 5 Ag of each heparin were dried down, resuspended in 100 mmol L sodium acetate, 0.1 mmol L calcium acetate pH 7.0 ; , and incubated with 5 milliunits each of heparin lyases I, II, and III for 18 hours at 37jC. Samples were boiled for 2 minutes and run through a prewashed Microcon 10 filter. Samples were then dried down, resuspended in MilliQ water, and separated by high-performance liquid chromatography HPLC ; on a Dionex ProPac PA1 anion exchange column using MilliQ water pH 3.5 ; with a sodium chloride gradient of 50 to 1, 000 mmol L over 60 minutes. Postcolumn derivatization with fluorescence detection was achieved by mixing 2-cyanoacetamide 1% ; with 250 mmol L NaOH in the eluent stream using an Eldex dual-channel pump. The eluent was then passed through an Eppendorf TC-40 reaction coil heated to 130jC, followed by a cooling bath, and then to a Jasco fluorescence detector set at an excitation of 346 nm and an emission of 410 nm. The sensitivity of this method is f5 pmol 50 ; . Heparin sizing. A TosoHaas TSKG2000SW HPLC column was run at 0.4 mL min in 10 mmol L KH2PO4, 0.5 mol L NaCl, and 0.2% Zwittergent Calbiochem, San Diego, CA ; . The void volume was determined using blue dextran. Cytidine monophosphate 0.5 Ag ; was spiked into all samples for use as an internal control to mark the included volume. Two different lots of each heparin were evaluated. Each sample was brought up to 10 total volume with MilliQ water. UV absorbance was monitored throughout the 45-minute runs at a wavelength of 206 nm. In an additional run, a larger aliquot of Tinzaparin 19.5 AL Tinzaparin and 2.5 Ag cytidine monophosphate marker ; was run on the.

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JAMA. 1999; 282: 1240-1246 Author Affiliations and Financial Disclosures are listed at the end of this article. CorrespondingAuthorandReprints: FrederickG.Hayden, jama MD, University of Virginia Health Sciences Center, Department of Internal Medicine, Box 473, Charlottesville, VA 22908 e-mail: fgh virginia.
HPAEC-PAD oligosaccharide mapping The PNGase F digestions were diluted to 200 #41 with ddH, O the PNGase F digestion of the total sRfBP tryptic peptides was rehydrolysed in 200 , ul of ddH20 ; , and 50 , ul was separated on a CarboPac-PA 100 column 0.4 cm x 25 cm; Dionex ; with a gradient of sodium acetate 50-250 mm in 60 min ; and a constant concentration of NaOH 100 mM ; after an initial 4 min at 50 mmsodium acetate and a constant concentration of NaOH 100 mM ; . Oligosaccharides were detected with the gold electrode and a detector setting of 100 nA full scale ; . Tentative identification of oligosaccharides Table 1 ; was made using oligosaccharide standards. A 150 u1 sample of each neuraminidase digestion was separated with the same gradient and dirithromycin.
REFERENCES 1. Chinese Traditional Medicine; People's Medical Publishing House, 2001. 2. Pharmacopoeia Commission of the Ministry of Public Health. Chinese Pharmacopoeia 2005, Part I. Chemical Industrial Press; Beijing, China, 2005. 3. Chinese Traditional Medical Chemistry. Shanghai Scientific & Technical Publishers; Shanghai, China, 2002. 4. Harkey, M. R.; Gary L Henderson, G. L.; Gershwin, M. E.; Stern, J. S.; Hackman, R. M. Variability in Commercial Ginseng Products: an Analysis of 25 Preparations. Am. J. of Clin. Nutr. 2001, 73, 11011106. Towards Efficient Extraction of Notogenseng Saponins from Cultured Cells of Panax notoginseng. Biochem. Eng. J. 2004, 18 2 ; , 115-120. 6. Dionex Corporation. Extraction of Chlorinated Pesticides Using Accelerated Solvent Extraction ASE Application Note 320, LPN 0637. Sunnyvale, CA, 2004. 7. Dionex Corporation. Rapid Extraction and Determination of Arsenicals in Fish Tissue and Plant Material Using Accelerated Solvent Extraction ASE Application Note 355, LPN 1782. Sunnyvale, CA, 2006.

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