Serum freezing temperature

Scholander and co-workers (1953, 1957 Gordon et al., 1962) made some of the initial significant observations on freezing resistances in fishes that could be interpreted on a molecular basis. They reported that the blood sera of Arctic fish had lower freezing temperatures than did the blood seras of fish not adapted to the cold. But, most important, they observed that the substances in the serum that helped to lower the freezing temperature were not due to salts, and that on chemical fractionation of the serum these substances were found in the fraction soluble in trichloroacetic acid. Other workers (Lievestad, 1965) also conducted related studies but in a more physiological vein. 

From Table I it can be seen that fish serum from cold ocean waters may have freezing temperatures as low as — 1.4°C, but not as low as those containing the antifreeze glycoproteins. The fish with freezing temperatures only as low as — 1.4°C were found in the Arctic south of the iceberg latitudes. The polar cod, containing the antifreeze glyco-... 

The effect of freezing and thawing must be distinguished from the effect of storage at freezing temperatures. Serum alkaline phosphatase... 

Anomalous Depression of the Freezing Temperature by Blood-Serum Proteins of Fishes... 

Figure 8. Photomicrographs of frog erythrocytes in serum during the course of slow freezing from -1.5 °C to -10 °C. Note that the cells are confined to the channels of unfrozen solution between the ice crystals (I), that the channels decrease in diameter with decreasing temperature, and that the cells shrink. (From Rapatz el al., 1966.)...

Greiff [3.24] studied the stability of purified influenza virus of the strain PR 8 in physiological NaCl solution with calcium lactobionate and human serum albumin (each 1 % in the solution). The freezing rate was approx. 1 °C/min down to -30 °C. During the freeze drying, the product temperature was raised in 12 to 16 h from -30 °C to 0 °C and the product was dried at this temperature. After 24 h, the first 145 vials were removed, and additional vials after intervals of 24 h each. The residual moisture content was 3.0,2.0,1.5, 1.0 and 0.5 %. The stability of the freeze dried virus (expressed in days during which the titer of the infectivity decreased by a factor of 10 was most unfavorable at 0.4 % and 3.2 % RM, (4 and 7 days respectively at +10 °C) and best at 1.7 % RM 145 days or more than 1000 days at -10 °C. 

The freeze drying of antibiotics and blood serum have largely represented the beginning of industrial lyophilization. Neumann [3.34] wrote in 1952 The (freeze drying) temperature for the older, not well purified Penicillin preparations had to be kept surprisingly low. It could not exceed -25 °C or -40 °C and later on Today Peniciilin is manufactured as crystals without the need of freeze drying . 

Cohn, and their associates in Harvard during World War II in their efforts to isolate serum proteins for clinical use. Traces of the organic solvents were removed during the freeze-drying of the different preparations. In the U.K. plasma fibrinogen was obtained by Kekwick and his colleagues by low-temperature precipitation with ether. Serum proteins acquired by these chemical methods were mixtures. 

Enzymes have different degrees of stability after their collection. Alkaline phosphatase demonstrates up to 10% increased activity after a few hours at room temperature (B15). Most enzymes are not stable at room temperature, but can be preserved in the refrigerator for short periods or in the deep freeze for relatively long times. In Table 4 are tabulated the reported stabilities of many serum enzymes. It must be realized that the problem of enzyme stability is complicated by the fact that the isoenzymes of a particular enzyme may have different stabilities and that specimens with high activities may react differently than those with normal activities (KIO). Although it is indicated in Table 4 that serum... 

Fish living in Arctic and Antarctic waters may encounter temperatures as low as -1.9°C. The freezing point depression provided by dissolved salts and proteins in the blood is insufficient to protect the fish from freezing. As winter approaches, they synthesize and accumulate in their blood serum a series of eight or more special antifreeze proteins.a d One type of antifreeze glycoprotein from winter flounder contains the following unit repeated 17-50 times. 

The stability of etodolac in equine serum and urine was studied under various temperatures (freezing, refrigerated, and room temperatures) for up to 45 days [32]. Etodolac was stable in urine and serum under frozen condition for 45 days, at refrigerated temperature for 30 days (7 days for urine). At room temperature, etodolac was stable in serum for up to 7 days, whereas it was stable in urine for 2 days. Etodolac serum and urine samples also showed stability through 5 freeze-thaw cycles. 

After the animal has been bled, the blood is allowed to dot by standing at room temperature for 1-2 h. The blood is centrifuged carefully at 5,000 g, avoiding lysis of the erythrocytes, to separate the serum and dotted fibrin factions. This antibody fraction can be stored at -80 °C for years without loss of activity. However, it is recommended that before aliquoting and freezing, the complement system should be inactivated, because this can interfere with many immunochemical reactions. Inactivation is carried out by simply heating to 56 °C for 10-20 min. 

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