Lloyd’s reagent

Subsequently, Howell improved the separation of heparin from protein by introducing the use of Lloyd s reagent (aluminum silicate) in acetic acid. The final stage then involved the precipitation of the heparin with excess barium hydroxide. Howell claimed that his heparin preparation con-... 

Beef liver (or lung) was minced and then autolyzed for twenty-four hours before extraction with an alkaline solution saturated with ammonium sulfate. Protein was precipitated by warming the extract, and the heparin-protein complex was precipitated from the supernatant liquor on acidification. Extraction of the complex with ethanol removed fatty material, and tryptic digestion removed most of the protein. The heparin was precipitated with ethanol, redissolved in warm alkaline solution to destroy trypsin, and reprecipitated with acetone. This material, crude heparin, was isolated in a yield of 15-50 g. per 100 lb. of animal tissue. In a later paper," the purification of crude heparin by fractionation successively with Lloyd s reagent, cadmium chloride, and acetone, was described. The purified heparin w-as 100 times as active as the crude material. Scott and Charles" reported the presence of nitrogen... 

A few years later, Jorpes confirmed much of the work done liy Charles and Scott. Heparin was purified by their procedure and, after. several successive treatments with Lloyd s reagent, the product was found to contain 1.64% of nitrogen. The presence of sulfur (as both bound and free sulfate), inorganic ash, uronic acid, hexosamine, and acetic acid was reported. Electrodialysis of the purified heparin removed free sulfate, and afforded a highly active preparation which could be precipitated as an amorphous brucine salt. ... 

Preparation of Extracts. Leffler (L2) and others have previously used isopropanol as a lipid solvent for determining cholesterol. Connerty et al. (C6) and Lofland (L4) also showed that triglycerides and phospholipids are extracted quantitatively. Under our extraction conditions, complete recovery of these lipid fractions is obtained at a serum-to-solvent ratio of 1 10 through 1 25. We routinely use 1 20 dilution (0.5 ml of sera+ 9.5 ml of isopropanol). We find that the addition of Lloyd s reagent removes bilirubin and other chromogenic material present in serum, as well as approximately 80% of the phospholipids present. 

Screw-capped culture tubes (16 X 150 mm) with Teflon-lined caps are used for carrying out the extractions. All tubes contain approximately 200 mg of Lloyd s reagent. Isopropanol (9.5 ml) is placed in tubes for sera, while tubes for standards contain 9.0 ml of isopropanol plus 0.5 ml of 0.9% NaCl. The contents of the tubes are mixed on a Vortex mixer, while adding 0.5 ml of serum or standard. Mixing is continued for 15 seconds. Tubes are capped and centrifuged, and the supernatant is decanted into another tube that is kept capped until used. 

Proteins and other impurities can be removed successfully by adsorption on zinc hydroxide (130), on Lloyd s reagent (130,161), on kaolin (88,130), on bentonite, and on magnesol (188). It must be realized, however, that an excess of these reagents will adsorb hyaluronic acid as well (130). 

Isolation from Synovial Fluid. The material obtained from the tarso-tibial joint of cattle or horses is diluted with 1 to 3 volumes of water and the hyaluronate and proteins present are precipitated by adding acetic acid to a concentration of 0.2%. Hyaluronic acid is isolated from the mucin precipitate either by solvent purification (10,88,133) or by enzymic digestion of the proteins with trypsin (161,163,168) or pancreatin (99). Digestion is considered complete when an aliquot does not give a mucin clot with acetic acid (168). The digest is purified by McClean s alcohol precipitation (107), by dialysis, or by adsorption on Lloyd s reagent (99, 161,168). Hyaluronate from synovial fluid frequently contains appreciable amounts of phosphorus. 

The minced tissue, cornea, nucleus pulposus or cartilage, was first treated with proteolytic enzymes, pepsin, tiypsin, or papain. Further removal of the protein, after pepsin treatment, was effected according to Sevag s procedure, or by adsorption on Lloyd s reagent. The polysaccharide mixture vras then fractionated by alcohol precipitation, or by alcohol elution of the barium salt on a cellulose column, or by passage through Dowex-i or Sephadex columns. 

---