therapy until an oral anticoagulant e.g. warfarin takes effect. the american college of chest physicians publishes clinical guidelines on heparin dosing. natural degradation or clearance unfractionated heparin has a half-life of about one to two hours after infusion, whereas low-molecular-weight heparin's half-life is about four times longer. lower doses of heparin have a much shorter half-life than larger ones. heparin binding to macrophage cells is internalized and depolymerized by the macrophages. it also rapidly binds to endothelial cells, which precludes the binding to antithrombin that results in anticoagulant action. for higher doses of heparin, endothelial cell binding will be saturated, such that clearance of heparin from the bloodstream by the kidneys will be a slower process. chemistry heparin structure ball-and-stick model of heparin native heparin is a polymer with a molecular weight ranging from 3 to 30 kda, although the average molecular weight of most commercial heparin preparations is in the range of 12 to 15 kda. heparin is a member of the glycosaminoglycan family of carbohydrates (which includes the closely related molecule heparan sulfate) and consists of a variably sulfated repeating disaccharide unit. the main disaccharide units that occur in heparin are shown below. the most common disaccharide unit* (see below) is composed of a 2-o-sulfated iduronic acid and 6-o-sulfated, n-sulfated glucosamine, idoa(2s)-glcns(6s). for example, this makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa. not shown below are the rare disaccharides containing a 3-o-sulfated glucosamine (glcns(3s,6s)) or a free amine group (glcnh3+). under physiological conditions, the ester and amide sulfate groups are deprotonated and attract positively charged counterions to form a heparin salt. heparin is usually administered in this form as an anticoagulant. idoa(2s)-glcns(6s)* idoa(2s)-glcns idoa-glcns(6s) glca-glcnac glca-glcns idoa-glcns glca = β-d-glucuronic acid, idoa = α-l-iduronic acid, idoa(2s) = 2-o-sulfo-α-l-iduronic acid, glcnac = 2-deoxy-2-acetamido-α-d-glucopyranosyl, glcns = 2-deoxy-2-sulfamido-α-d-glucopyranosyl, glcns(6s) = 2-deoxy-2-sulfamido-α-d-glucopyranosyl-6-o-sulfate one unit of heparin (the "howell unit") is an amount approximately equivalent to 0.002 mg of pure heparin, which is the quantity required to keep 1 ml of cat's blood fluid for 24 hours at 0 °c. three-dimensional structure the three-dimensional structure of heparin is complicated because iduronic acid may be present in either of two low-energy conformations when internally positioned within an oligosaccharide. the conformational equilibrium is influenced by sulfation state of adjacent glucosamine sugars. nevertheless, the solution structure of a heparin dodecasaccharide composed solely of six glcns(6s)-idoa(2s) repeat units has been determined using a combination of nmr spectroscopy and molecular modeling techniques. two models were constructed, one in which all idoa(2s) were in the 2s0 conformation (a and b below), and one in which they are in the 1c4 conformation (c and d below). however, no evidence suggests that changes between these conformations occur in a concerted fashion. these models correspond to the protein data bank code 1hpn. two different structures of heparin in the image above: a = 1hpn (all idoa(2s) residues in 2s0 conformation) jmol viewer b = van der waals radius space filling model of a c = 1hpn (all idoa(2s) residues in 1c4 conformation) jmol viewer d = van der waals radius space filling model of c in these models, heparin adopts a helical conformation, the rotation of which places clusters of sulfate groups at regular intervals of about 17 angstroms (1.7 nm) on either side of the helical axis. depolymerization techniques either chemical or enzymatic depolymerization techniques or a combination of the two underlie the vast majority of analyses carried out on the structure and function of heparin and heparan sulfate (hs). enzymatic the enzymes traditionally used to digest heparin or hs are naturally produced by the soil bacterium pedobacter heparinus (formerly named flavobacterium heparinum). this bacterium is capable of using either heparin or hs as its sole carbon and nitrogen source. to do so, it produces a range of enzymes such as lyases, glucuronidases, sulfoesterases, and sulfamidases. the lyases have mainly been used in heparin/hs studies. the bacterium produces three lyases, heparinases i (ec 4.2.2.7), ii (no ec number assigned) and iii (ec 4.2.2.8) and each has distinct substrate specificities as detailed below. heparinase enzymesubstrate specificity heparinase iglcns(±6s)-idoa(2s) heparinase iiglcns/ac(±6s)-idoa(±2s) glcns/ac(±6s)-glca heparinase iiiglcns/ac(±6s)-glca/idoa (with a preference for glca) ua(2s)-glcns(6s) the lyases cleave heparin/hs by a beta elimination mechanism. this action generates an unsaturated double bond between c4 and c5 of the uronate residue. the c4-c5 unsaturated uronate is termed δua or ua. it is a sensitive uv chromophore (max absorption at 232 nm) and allows the rate of an enzyme digest to be followed, as well as providing a convenient method for detecting the fragments produced by enzyme digestion. chemical nitrous acid can be used to chemically depolymerize heparin/hs. nitrous acid can be used at ph 1.5 or at a higher ph of 4. under both conditions, nitrous acid effects deaminative cleavage of the chain. idoa(2s)-aman: the anhydromannose can be reduced to an anhydromannitol at both 'high' (4) and 'low' (1.5) ph, deaminative cleavage occurs between glcns-glca and glcns-idoa, albeit at a slower rate at the higher ph. the deamination reaction, and therefore chain cleavage, is regardless of o-sulfation carried by either monosaccharide unit. at low ph, deaminative cleavage results in the release of inorganic so4, and the conversion of glcns into anhydromannose (aman). low-ph nitrous acid treatment is an excellent method to distinguish n-sulfated polysaccharides such as heparin and hs from non n-sulfated polysaccharides such as chondroitin sulfate and dermatan sulfate, chondroitin sulfate and dermatan sulfate not being susceptible to nitrous acid cleavage. detection in body fluids current clinical laboratory assays for heparin rely on an indirect measurement of the effect of the drug, rather than on a direct measure of its chemical presence. these include activated partial thromboplastin time (aptt) and antifactor xa activity. the specimen of choice is usually fresh, nonhemolyzed plasma from blood that has been anticoagulated with citrate, fluoride, or oxalate. other functions blood specimen test tubes, vacutainers, and capillary tubes that use the lithium salt of heparin (lithium heparin) as an anticoagulant are usually marked with green stickers and green tops. heparin has the advantage over edta of not affecting levels of most ions. however, the concentration of ionized calcium may be decreased if the concentration of heparin in the blood specimen is too high. heparin can interfere with some immunoassays, however. as lithium heparin is usually used, a person's lithium levels cannot be obtained from these tubes; for this purpose, royal-blue-topped (and dark green-topped) vacutainers containing sodium heparin are used. heparin-coated blood oxygenators are available for use in heart-lung machines. among other things, these specialized oxygenators are thought to improve overall biocompatibility and host homeostasis by providing characteristics similar to those of native endothelium. the dna binding sites on rna polymerase can be occupied by heparin, preventing the polymerase from binding to promoter dna. this property is exploited in a range of molecular biological assays. common diagnostic procedures require pcr amplification of a patient's dna, which is easily extracted from white blood cells treated with heparin. this poses a potential problem, since heparin may be extracted along with the dna, and it has been found to interfere with the pcr reaction at levels as low as 0.002 u in a 50 μl reaction mixture. heparin has been used as a chromatography resin, acting as both an affinity ligand and an ion exchanger. its polyanionic structure can mimic nucleic acids like dna and rna, making it useful for purification of nucleic acid-binding proteins including dna and rna polymerases and transcription factors. heparin's specific affinity for vsv-g, a viral envelope glycoprotein often used to pseudotype retroviral and lentiviral vectors for gene therapy, allows it to be used for downstream purification of viral vectors. heparin is being trialed in a nasal spray form as prophylaxis against covid-19 infection. furthermore, its reported from trials that due to anti-viral, anti-inflammatory and its anti-clotting effects its inhalation could improve at a 70% rate on patients that were actively struck by a covid-19 infection. society and culture contamination recalls considering the animal source of pharmaceutical heparin, the numbers of potential impurities are relatively large compared with a wholly synthetic therapeutic agent. the range of possible biological contaminants includes viruses, bacterial endotoxins, transmissible spongiform encephalopathy (tse) agents, lipids, proteins, and dna. during the preparation of pharmaceutical-grade heparin from animal tissues, impurities such as solvents, heavy metals, and extraneous cations can be introduced. however, the methods employed to minimize the occurrence and to identify and/or eliminate these contaminants are well established and listed in guidelines and pharmacopoeias. the major challenge in the analysis of heparin impurities is the detection and identification of structurally related impurities. the most prevalent impurity in heparin is dermatan sulfate (ds), also known as chondroitin sulfate b. the building-block of ds is a disaccharide composed of 1,3-linked n-acetyl galactosamine (galn) and a uronic acid residue, connected via 1,4 linkages to form the polymer. ds is composed of three possible uronic acid (glca, idoa or idoa2s) and four possible hexosamine (galnac, gal- nac4s, galnac6s, or galnac4s6s) building-blocks. the presence of iduronic acid in ds distinguishes it from chrondroitin sulfate a and c and likens it to heparin and hs. ds has a lower negative charge density overall compared to heparin. a common natural contaminant, ds is present at levels of 1–7% in heparin api, but has no proven biological activity that influences the anticoagulation effect of heparin. in december 2007, the us food and drug administration (fda) recalled a shipment of heparin because of bacterial growth (serratia marcescens) in several unopened syringes of this product. s. marcescens can lead to life-threatening injuries and/or death. 2008 recall due to adulteration in drug from china main article: 2008 chinese heparin adulteration in march 2008, major recalls of heparin were announced by the fda due to contamination of the raw heparin stock imported from china. according to the fda, the adulterated heparin killed nearly 80 people in the united states. the adulterant was identified as an "over-sulphated" derivative of chondroitin sulfate, a popular shellfish-derived supplement often used for arthritis, which was intended to substitute for actual heparin in potency tests. according to the new york times: "problems with heparin reported to the agency include difficulty breathing, nausea, vomiting, excessive sweating and rapidly falling blood pressure that in some cases led to life-threatening shock". use in homicide in 2006, petr zelenka, a nurse in the czech republic, deliberately administered large doses to patients, killing seven, and attempting to kill ten others. overdose issues in 2007, a nurse at cedars-sinai medical center mistakenly gave the 12-day-old twins of actor dennis quaid a dose of heparin that was 1,000 times the recommended dose for infants. the overdose allegedly arose because the labeling and design of the adult and infant versions of the product were similar. the quaid family subsequently sued the manufacturer, baxter healthcare corp., and settled with the hospital for $750,000. prior to the quaid accident, six newborn babies at methodist hospital in indianapolis, indiana, were given an overdose. three of the babies died after the mistake. in july 2008, another set of twins born at christus spohn hospital south, in corpus christi, texas, died after an accidentally administered overdose of the drug. the overdose was due to a mixing error at the hospital pharmacy and was unrelated to the product's packaging or labeling. as of july 2008, the exact cause of the twins' death was under investigation. in march 2010, a two-year-old transplant patient from texas was given a lethal dose of heparin at the university of nebraska medical center. the exact circumstances surrounding her death are still under investigation. production pharmaceutical-grade heparin is derived from mucosal tissues of slaughtered meat animals such as porcine (pig) intestines or bovine (cattle) lungs. advances to produce heparin synthetically have been made in 2003 and 2008. in 2011, a chemoenzymatic process of synthesizing low molecular weight heparins from simple disaccharides was reported. research as detailed in the table below, the potential is great for the development of heparin-like structures as drugs to treat a wide range of diseases, in addition to their current use as anticoagulants. disease states sensitive to heparinheparin's effect in experimental modelsclinical status acquired immunodeficiency syndromereduces the ability of human immunodeficiency virus types 1 and 2 to adsorb to cultured t4 cells.– adult respiratory distress syndromereduces cell activation and accumulation in airways, neutralizes