Hemolysis, erythrocytic, assay

There is no clinical disease state that is pathognomonic for lead exposure. The neurotoxic effects and hematopoietic effects of lead are well recognized. The primary biomarkers of effect for lead are EP, ALAD, basophilic stippling and premature erythrocyte hemolysis, and presence of intranuclear lead inclusion bodies in the kidneys. Of these, activity of ALAD is a sensitive indicator of lead exposure (Hemberg et al. 1970 Morris et al. 1988 Somashekaraiah et al. 1990 Tola et al. 1973), but the assay can not distinguish between moderate and severe exposure (Graziano 1994). Sensitive, reliable, well-established methods exist to monitor for these biomarkers however, they are not specific for lead exposure. Therefore, there is a need to develop more specific biomarkers of effect for lead. Recent data... 

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

The serum should be yellowish. A reddish color is indicative of erythrocytes lysis (hemolysis) which may interfere with clinical chemistry assays based on colorimetric values. If this happens, you may need to adjust the speed at which the blood is collected and processed or other steps that may cause sheer and red blood cell lysis. 

There is also increased erythrocyte hemolysis, which responds to synthetic antioxidants or selenium. The sensitivity of erythrocytes to chemically induced hemolysis can be used both as a biological assay of vitamin E in... 

Both are abundant in skeletal muscle, myocardium, liver, and erythrocytes, so that hemolysis must be avoided, and in serum they may be assayed spectrophotometrically by their conversion of phosphate-buffered pyruvate to lactate (R6, W16) or oxalacetate to malate (S25) at the expense of added NADH2, when the rate of decrease of optical density at 340 m x thus measmes the serum activities of the respective enzymes. Recently, however, the reverse reaction has been found best for serum lactic dehydrogenase assay (A2a). In conventional spectrophotometric units the normal ranges are 100-600 units per ml for lactic dehydrogenase (W16) and 42-195 xmits per ml for malic dehydrogenase (S25) as before, one conventional spectrophotometric unit per ml = 0.48 pmoles/ minute/liter (W13). 

Other pitfalls in correct diagnosis of glycolytic enzyme deficiencies include the red cell age dependency of enzymes such as PK, HK, and G6PD. The measurement of these enzymes simultaneously can give an idea about red cell age and relative deficiencies. Many patients suffering from severe hemolysis have already received blood transfusions. When this occurs, interpreting results from red cell enzyme aissays must be done with great care, since the. presence of donor erythrocytes wdl obscme any deficiencies. In addition, some mutant enzymes display a normal activity in vitro, while in vivo severe hemolysis can occur. More sophisticated assays to measure, for example, heat instability and kinetics have to be used in those cases. 

Erythrocyte GSH concentration is diminished in many people who have defects in the hexose monophosphate or GSH synthesis pathways. The GSH stability test, originally devised to permit identification of people susceptible to hemolysis from primaquine (later shown to be the result of G6PD deficiency), still remains a useful "stress test of the intactness of these closely hnked pathways. Because deficiencies of GSH synthetase and y-glutamyl cysteine synthetase are rare disorders, it is not practical for clinical laboratories to contemplate assays for these enzymes unless results of the easily performed GSH stability test are abnormal. 

Serum, heparinized plasma, whole blood, sweat, urine, feces, or gastrointestinal fluids may be assayed for Nah Timed collections of urine, feces, or gastrointestinal fluids are preferred to allow comparison of values with reference intervals and to determine rates of electrolyte loss. Serum, plasma, and urine may be stored at 2 C to 4 C or frozen. Erythrocytes contain only one tenth of the Na" present in plasma, so hemolysis does not cause significant errors in serum or plasma Na values. Lipemic samples should be ultracen-trifuged and the infranatant analyzed unless a direct ISE is used. 

Samples for this assay may be cell supernatant from cell culture experiments, cell lysates, tissue lysates, or biological fluids such as serum or plasma. High degree of hemolysis in serum or blood in tissue samples interferes with the measurement due to the presence of erythrocyte acid phosphatase. 

The normal lactate concentration in blood is between 1.2 and 2.7 mmol/1. For accurate lactate determination hemolysis of the sample is required to account for the (low) lactate content of erythrocytes. On the other hand, the glycolytic reactions in the sample have to be efficiently and rapidly inhibited in order to avoid lactate formation. Therefore the best-suited sample material is deproteinized blood however, the time period inevitably required for its preparation prevents rapid lactate assay. That is why the study of blood lactate sensors focuses not only on the sensor itself but also on the rapid pretreatment of blood samples. 

Erythrocyte hemolysis. Hemolysis experiments were performed in PBS using static method, as previously described [2], The degree of hemolysis due to sample activity was calculated as the hemolytic index %H = (Hb - Hbo/Hb,ot) 100%, where Hb is the total amount of released hemoglobin in the assayed sample, Hbo the amount released due to basal hemolysis (200pl erythrocytes incubated with 50pl PBS), and Hb,ot the total amount of released hemoglobin in fully disrupted erythrocytes (0.2 ml erythrocytes in 19.8 ml H2O MilU Q). 

This enzymatic activity was assayed by indirect hemolysis as described by Guttierrez et al [5] with a slight modification instead of sheep erythrocytes, mice red blood cells were employed. Previous experiments have shown that this little change had no effect on the assay sensitivity [8]. 0.3pg of each venom were incubated with either phosphate buffered saline, pure or 1 10 diluted antiserum and applied to 2mm wells punched on a glass plate covered with a mixture of 1.2% fresh washed mice erythrocytes, 1.2% of 1 4 diluted egg yolk and lOmM calcium chloride in 0.8% agarose medium. Phosphate buffered saline was used as negative control. The diameter of the hemolysis haloes were measured after 24 hours incubation at 37°C. 

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