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Both aminotransferases are highly concentrated in the liver. AST is also diffusely represented in the heart, skeletal muscle, kidneys, brain and red blood cells, and ALT has low concentrations in skeletal muscle and kidney;21 an increase in ALT serum levels is, therefore, more specific for liver damage. In the liver, ALT is localized solely in the cellular cytoplasm, whereas AST is both cytosolic (20% of total activity) and mitochondrial (80% of total activity).22 Zone 3 of the hepatic acinus has a higher concentration of AST, and damage to this zone, whether ischemic or toxic, may result in greater alteration to AST levels. Aminotransferase clearance is carried out within the liver by sinusoidal cells.23 The half-life in the circulation is about 47 hours for ALT, about 17 hours for total AST and, on average, 87 hours for mitochondrial AST.4
Despite these ambiguities, the magnitude and rate of change of aminotransferase alteration may provide initial insight into a differential diagnosis. Very high aminotransferase levels (> 75 times the upper reference limit) indicate ischemic or toxic liver injury in more than 90% of cases of acute hepatic injury, whereas they are less commonly observed with acute viral hepatitis.4 In ischemic or toxic liver injury, AST levels usually peak before those of ALT because of the enzyme's peculiar intralobular distribution.27,28,29 Zone 3 of the acinus is more vulnerable to both hypoxic (hepatocytes are exposed to an already oxygen-poor milieu) and toxic (hepatocytes are richer in microsomal enzymes) damage. Furthermore, in ischemic injury aminotransferase levels tend to decrease rapidly after peaking (Fig. 3). In about 80% of patients with ischemic injury, the serum bilirubin level is lower than 34 μmol/L, and lactate dehydrogenase (LDH), a marker of ischemic damage, may reach very high concentrations (ALT/LDH ratio < 1).28,29,30 It is important to stress that a decrease in aminotransferase levels alone after a marked increase does not have prognostic meaning, since both resolution and massive hepatic necrosis may draw a similar biochemical picture. In this setting, patients with high bilirubin serum levels and prolonged prothrombin time should be closely monitored for the risk of hepatic failure.
Unconjugated bilirubin may increase because of augmented bilirubin production or decreased hepatic uptake or conjugation or both (Table 3). In adults, the most common conditions associated with unconjugated hyperbilirubinemia are hemolysis and Gilbert's syndrome.78 Hemolysis can be ruled out by measuring hemoglobin serum levels, reticulocyte count, and haptoglobin levels. Gilbert's syndrome is determined by a variety of genetic defects in UDP-glucuronyltransferase that affect about 5% of the population.79 In these subjects serum indirect bilirubin usually does not exceed 68 μmol/L, and the remainder of liver chemistry tests and liver ultrasound are unremarkable.80 Although a series of tests has been proposed to confirm the diagnosis, this condition is usually diagnosed on a clinical basis alone, and the patient should be reassured of the benign nature of this enzymatic alteration.80 Other, less frequent causes of unconjugated hyperbilirubinemia include reabsorption of large hematomas and ineffective erythropoiesis.24
In healthy people, conjugated bilirubin is virtually absent from serum mainly because of the rapid process of bile secretion.1 Levels increase when the liver has lost at least half of its excretory capacity. Therefore, the presence of increased conjugated bilirubin is usually a sign of liver disease. Conjugated hyperbilirubinemia (usually < 34 μmol/L) and concomitant, markedly elevated aminotransferase levels may suggest acute viral hepatitis or toxic or ischemic liver injury. Furthermore, this biochemical picture can be the presenting feature of autoimmune hepatitis.40,41,42 On the other hand, a purely cholestatic picture, with conjugated hyperbilirubinemia, an increase in ALP levels and a negligible increase in aminotransferase levels, may be present in cholestatic drug reactions.16,68 Sometimes, the same biochemical picture may be present in the late presentation of previously unrecognized autoimmune cholestatic diseases (primary biliary cirrhosis, primary sclerosing cholangitis). In these patients, the presence of other signs of chronic liver disease may facilitate diagnosis.62,69,70
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The food additive BHA (butylated hydroxyanisole) alone or in combination with other antioxidants permitted in food for human consumption in this subpart B may be safely used in or on specified foods, as follows:
Microcapsules may be safely used for encapsulating discrete particles of flavoring substances that are generally recognized as safe for their intended use or are regulated under this part, in accordance with the following conditions:
All glassware should be scrupulously cleaned to remove all organic matter such as oil, grease, detergent residues, etc. Examine all glassware, including stoppers and stopcocks, under ultraviolet light to detect any residual fluorescent contamination. As a precautionary measure, it is recommended practice to rinse all glassware with purified isooctane immediately before use. No grease is to be used on stopcocks or joints. Great care to avoid contamination of petroleum naphtha samples in handling and to assure absence of any extraneous material arising from inadequate packaging is essential. Because some of the polynuclear hydrocarbons sought in this test are very susceptible to photo-oxidation, the entire procedure is to be carried out under subdued light.
n-Hexadecane, 99 percent olefin-free. Dilute 1.0 milliliter of n-hexadecane to 25 milliliters with isooctane and determine the absorbance in a 5-centimeter cell compared to isooctane as reference between 280-400 mµ. The absorbance per centimeter path length shall not exceed 0.00 in this range. Purify, if necessary, by percolation through activated silica gel or by distillation.
Determination of ultraviolet absorbance. Add a 25-milliliter aliquot of the hydrocarbon solvent together with 1 milliliter of hexadecane to the 125-milliliter Erlenmeyer flask. While flushing with nitrogen, evaporate to 1 milliliter on a steam bath. Nitrogen is admitted through a 8±1-milliliter outer-diameter tube, drawn out into a 2±1-centimeter long and 1±0.5-millimeter inner-diameter capillary tip. This is positioned so that the capillary tip extends 4 centimeters into the flask. The nitrogen flow rate is such that the surface of the liquid is barely disturbed. After the volume is reduced to that of the 1 milliliter of hexadecane, the flask is left on the steam bath for 10 more minutes before removing. Add 10 milliliters of purified isooctane to the flask and reevaporate the solution to a 1-milliliter volume in the same manner as described above, except do not heat for an added 10 minutes. Repeat this operation twice more. Let the flask cool.
Add 10 milliliters of methyl alcohol and about 0.3 gram of sodium borohydride. (Minimize exposure of the borohydride to the atmosphere; a measuring dipper may be used.) Immediately fit a water-cooled condenser equipped with a 24/40 joint and with a drying tube into the flask, mix until the sodium borohydride is dissolved, and allow to stand for 30 minutes at room temperature, with intermittent swirling. At the end of this time, disconnect the flask and evaporate the methyl alcohol on the steam bath under nitrogen until sodium borohydride begins to drop out of solution. Remove the flask and let it cool.
Add 6 milliliters of isooctane to the flask and swirl to wash the crystalline slurry. Carefully transfer the isooctane extract to a 250-milliliter separatory funnel. Dissolve the crystals in the flask with about 25 milliliters of distilled water and pour this also into the separatory funnel. Adjust the water volume in the separatory funnel to about 100 milliliters and shake for 1 minute. After separation of the layers, draw off the aqueous layer into a second 250-milliliter separatory funnel. Transfer the hydrocarbon layer in the first funnel to a 25-milliliter volumetric flask.
 As determined by procedure using potassium chromate for reference standard and described in National Bureau of Standards Circular 484, Spectrophotometry, U.S. Department of Commerce, (1949). The accuracy is to be determined by comparison with the standard values at 290, 345, and 400 millimicrons. The procedure is incorporated by reference. Copies of the material incorporated by reference are available from the Center for Food Safety and Applied Nutrition (HFS-200), Food and Drug Administration, 5001 Campus Dr., College Park, MD 20740, or available for inspection at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: _register/code_of_federal_regulations/ibr_locations.html.
 Copies are available from: AOAC INTERNATIONAL, 481 North Frederick Ave., suite 500, Gaithersburg, MD 20877, or examined at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: _register/code_of_federal_regulations/ibr_locations.html. 2b1af7f3a8