Hemoglobin removal

Isolation of hemoglobin, removal of water, followed by alkaline hydrolysis of adducts addition of 2,2 -dichloro-benzidine as internal standard extraction suing toluene containing 5% 2-propanol derivatization using HFBA. 

The half life for NO in cellular systems ranges from 5—30 seconds. Superoxide, hemoglobin, and other radical trapping agents remove NO after it has been formed. 

Phthalocyanines are excellent lubricants at temperatures of 149—343°C (191). Combinations with other lubricants, like grease, molybdenum, or tungsten sulfides, have found appHcations in the automotive industry or professional drilling equipment (192—195). Further uses include indicators for iron(Il), molybdenum(V), and uranium(IV) (196) or redox reactions (197), medical appHcations like hemoglobin replacements (198) or sterilisation indicators (199), or uses like in gas filters for the removal of nitrogen oxides from cigarette smoke (200). 

Toxicity. A 1% concn of the gas in air is lethal to rats in 1 hour, its effect being similar to C monoxide the LD50 in rats when injected intra-peritoneally is 8.2ml/kg (Ref 16). Earlier workers assumed that the toxicity of N trifluoride would be similar to H fluoride and that the latter would be formed by hydrolysis in body tissues (Ref 1). This has recently been shown to be erroneous, and that it is stable under physiological conds. The toxic effect is due to its ability to complex with the hemoglobin of the blood causing anoxia. This effect is reversible, and animals receiving a sublethal dose recover rapidly upon removal from contact with N trifluoride (Ref 14)... 

Many use the hexokinase procedure without protein precipitation. That this is a procedure which is not acceptable is illustrated in Table II where it is shown that unless hemoglobin is removed there will be interference in the results obtained. In severely hemolyzed blood, errors as high as 25% are not uncommon. 

Local administration of NO to the lungs has been shown to reverse pulmonary hypertension in animal models [103], importantly with no systemic side effects. This is likely to be as a result of surplus NO being removed as nitrosyl-hemoglobin [104]. Such advantages of gaseous NO were first reported in 1991 [105, 106]. In 1999 and 2001 NO gas was approved as a drug in the USA and European Union, for treating hypoxemic respiratory failure in infants [107]. 

Ultimately, over periods that may be very short, as in the case of volatile substances such as acetone or alcohol, or very long, as with substances such as the dyestuff aniline which latches on to the hemoglobin in the blood that normally carries oxygen, most chemicals will be removed from the body by the body s natural processes. As we have seen, however, there are exceptions, such as lead, that will stay in the bones forever. 

AmB formulations were dispersed in phosphate-buffered saline (PBS) at different concentrations (0.1 lOOpg/mL) and incubated for five minutes at 37°C. Freshly isolated human erythrocytes were then added to a final hematocrit of 2% and incubated at the same temperature for 30 minutes. After centrifugation, the supernatant was removed and the RBC pellet was lysed with sterile water. The hemoglobin remaining in the pellet was estimated from its absorption at 560 nm recorded with a spectrophotometer. The percentage hemolysis was calculated from the difference between the hemoglobin remaining in the test samples and the control incubated with PBS alone. 

Non-statistical successive binding of O2 and CO to the four heme centers of hemoglobin ( cooperativity ) has been thoroughly documented. It is difficult to test for a similar effect for NO since the equilibrium constants are very large ( 10 M ) and therefore difficult to measure accurately. It is found that the four successive formation rate constants for binding NO to hemoglobin are identical. In contrast, the rate constant for dissociation of the first NO from Hb(NO)4 is at least 80 times less than that for removal of NO from the singly bound entity Hb(NO). This demonstrates cooperativity for the system, and shows that it resides in the dissociation process. The thermodynamic implications of any kinetic data should therefore always be assessed. 

Haptoglobin binds hemoglobin in a 1 1 molar complex (two ap dimers per haptoglobin) (42). This complex is quickly removed from the circulation via a suicidal receptor-mediated endocytosis (43,44), which consequently depletes haptoglobin. Even a short bout of exercise can deplete haptoglobin (45), whose total plasma amount is equivalent to the hemoglobin in about 4.5 cm of red cells. 

Fig. 1. Overview of intravascular heme catabolism. Hemoglobin, myoglobin, and other heme proteins are released into the circulation upon cellular destruction, and the heme moiety is oxidized by O2 to the ferric form (e.g., methemoglobin and metmyoglobin). Haptoglobin can bind a substantial amount of hemoglobin, but is readily depleted. Ferric heme dissociates from globin and can be bound by albumin or more avidly by hemopexin. Hemopexin removes heme from the circulation by a receptor-mediated transport mechanism, and once inside the ceU heme is transported to heme oxygenase for catabolism.

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