Blood storage XXIV: red blood cell 2,3-DPG and ATP maintenance for six weeks in CPD-adenine with higher phosphate, pyruvate, and dihydroxyacetone

The individual and collective effects of various phosphate, pyruvate and dihydroxyacetone concentrations on 2,3-DPG and ATP maintenance during blood storage with CPD-adenine (0.25 mM), were studied. Phosphate concentrations ranged from 2 to 100 mM. Low concentations were best for 2,3-DPG maintenance during the first three weeks, after which there was no difference. ATP concentrations were better maintained by the highest phosphate concentrations in the first week. After the second week the lower concentrations of phosphate were better. With pyruvate 40 and 60 mM were the best for 2,3-DPG levels through six weeks of storage. ATP concentrations were poorest with high pyruvate. Maintenance of 2,3-DPG was above half normal for six weeks of storage in the 60, 80 and 100 mM DHA preservatives. ATP concentrations were best maintained in the preservative lacking DHA. Combinations of phosphate, pyruvate and DHA in concentrations which had been found to be effective when used individually were studied. Best maintenance of 2,3-DPG (above half normal levels) for six weeks was afforded by pyruvate, phosphate and DHA, and by pyruvate and DHA. ATP maintenance was best afforded by CPD-adenine alone and CPD-adenine with pyruvate and phosphate. Pyruvate alone maintained ATP less well and the pyruvate- DHA was worst. Intermediate in maintenance of ATP was the preservative containing pyruvate, phosphate and DHA.     

Blood preservation 35. Red cell 2,3-DPG and ATP maintained by DHA- ascorbate-phosphate

DHA (dihydroxyacetone, 60 mM) with ascorbic acid (d-ascorbate, 10 mM) kept 2,3-DPG concentrations above normal for six weeks. Levels of 2,3- DPG were below normal after four weeks with DHA alone and after two weeks with DHA-ascorbate-phosphate. As in previous studies, high phosphate concentrations decreased 2,3-DPG maintenance. ATP maintenance was best achieved with the following (in order of performance): DHA- phosphate (20 mM); DHA-phosphate (10 mM); the control, CPD-adenine preservative; Phosphate 20 mM; and DHA. DHA with ascorbate provides normal 2,3-DPG for six weeks. The adverse effects of DHA and DHA with ascorbate on ATP levels are modified by 10 mM phosphate.     

Refrigerated storage of lyophilized and rehydrated, lyophilized human red cells

Human red cells (RBCs) were collected in CPDA-1 and then freeze-dried in lyoprotective solution. The lyophilized RBCs were then stored at -20 degrees C for 7 days. At the end of the storage period, the lyophilized RBCs were rehydrated and washed in dextrose saline. The washed, reconstituted, lyophilized RBCs were resuspended in final wash solutions of ADSOL, CPDA-1, or a special additive solution containing glucose, citrate, phosphate, adenine, and mannitol, and then they were stored at 4 degrees C for an additional 7 days. The main purpose of this study was to determine whether human RBCs can be lyophilized in such a manner that normal metabolic, rheologic, and cellular properties are maintained during rehydration and subsequent storage in standard blood bank preservative solutions. Our results show that reconstituted, lyophilized RBCs maintained levels of ATP, 2,3 DPG, lactate, and cellular properties that are equal to or better than those in control nonlyophilized RBCs stored for a comparable period in CPDA-1. Reconstituted, lyophilized RBCs stored at 4 degrees C after rehydration also show better maintenance of ATP, 2,3 DPG, and lactate than do control RBCs stored in the same preservative solutions for comparable periods.     

Blood storage XXII. Improvement in red blood cell 2,3-DPG levels at six weeks by 20 mM PO4 in CPD-adenine-inosine

Inorganic phosphate has been known to assist red blood cell maintenance of ATP and in the presence of inosine to assist in the maintenance of 2,3-DPG. High concentrations of phosphate, while helping ATP maintenance, were found to be deleterious to 2,3-DPG maintenance in CPD- adenine preservatives. However, in the presence of inosine, concentrations of phosphate as high as 10 mM were advantageous to 2,3- DPG maintenance. The present study extends the observations on ATP and 2,3-DPG maintenance in CPD-adenine-inosine preservatives from the previous 10 mM to 20 mM phosphate. A high phosphate (20 mM) effect has been seen as improved maintenance of 2,3-DPG levels during the fifth and sixth weeks of storage of whole blood at 4C. This supports the previously reported observation of improved maintenance of 2,3-DPG in a 10 mM phosphate preservative. This is ten times the 2 mM phosphate concentration in CPD-adenine. In the low phosphate preservative (2 mM), 2,3-DPG maintenance is less than that in all of the higher phosphate preservatives after the second week of storage. ATP concentrations in this experiment show good maintenance throughout six weeks of storage.     

Blood preservation. XXIX. Pyruvate maintains normal red cell 2,3-DPG for six weeks of storage in CPD-adenine

Pyruvate was placed in experimental CPD-adenine (0.25 mM) blood preservative mixtures in four concentrations ranging from 40 to 320 mM. In the 320 mM pyruvate preservative, 2,3-DPG levels were elevated above normal for six weeks of whole blood storage at 4 C. The lower pyruvate concentrations maintained elevated or normal 2,3-DPG levels for less time: four weeks with 160 mM, two weeks with 80 mM, and one week or less with 40 mM or the control. ATP values were best maintained in the control. The higher pyruvate concentrations resulted in the most rapid decreases at ATP. However, even the 320 mM pyruvate did not cause ATP to fall below 2 microM/gm of Hb. The higher pyruvate concentrations produced and maintained a higher pH during storage. On the other hand, 2,3-DPG levels increased with pyruvate during the first week of storage when the pH was decreasing rapidly. This could be the result of its oxidation of NADH to NAD. The high pyruvate concentration which maintained elevated 2,3-DPG levels throughout the six weeks might be simulating the effect reported in pyruvate kinase-deficient red blood cells, in which blockage of glycolysis at that step is preventing 2,3- DPG catabolism.     

The effect of additives on red cell 2,3 diphosphoglycerate levels in CPDA preservatives

Forty-two chemical substances, chosen because they might influence red cell metabolism, were screened for effect on red cell adenosine triphosphate and 2,3 diphosphoglycerate (2,3 DPG) levels during storage in CPD or CPDA-1 at 4 degrees C. Of these substances, six appeared on initial screening to increase 2,3 DPG levels during storage; on repeated examination, four compounds, i.e., oxalate, glyoxalate, ethyl oxaloacetate, and L-phenylalanyl-L-alanine, consistently increased 2,3 DPG levels during storage. It was shown that glyoxalate was converted rapidly to oxalate in blood, presumably through the lactate dehydrogenase reaction. Ethyl oxaloacetate is known to hydrolyze, giving rise to oxalate. Thus, the effect of both glyoxalate and ethyl oxaloacetate can be explained by the formation of oxalate, a compound already known to increase 2,3 DPG levels. The effect of L-phenylalanyl-L-alanine remains to be explained, but it may be hydrolyzed to L-alanine and L-phenylalanine, both of which are thought to have the capacity to increase red cell 2,3 DPG levels by inhibiting pyruvate kinase activity.     

Blood preservation. XXXIV. DHA maintains 2,3-DPG and its adverse effect on ATP is reversed with extra phosphate

We have found that the addition of 10 mM inorganic phosphate to DHA in CPD-adenine maintains ATP levels at normal or higher than normal values for six weeks of storage. 2,3-DPG values are slightly lowered by the extra phosphate, but are still maintained at approximately half normal for four weeks by the DHA. The addition of a higher phosphate concentration, 20 mM, to DHA produced lower levels of ATP and 2,3-DPG than those observed with 10 mM phosphate, although both levels were better than in the CPD-adenine control. pH values in this experiment were lowest in the three preservatives containing DHA, probably indicating increased lactate production due to metabolism of this triose sugar, in addition to dextrose present in CPD.     

Preservation of Red Blood Cell 2,3‐Diphosphoglycerate in Stored Blood Containing Dihydroxyacetone

In a search for red blood cell metabolites which would preserve 2,3‐DPG during storage of blood, it was discovered that dihydroxyacetone (DHA) prolonged the maintenance of 2,3‐DPG levels for up to four weeks of storage, compared to about one week for presently used preservatives such as CPD. Four‐week preservation of 2,3‐DPG at normal levels was desired. CPD‐adenine was used as the starting point and a formulation having a pH of 7.0 and a DHA concentration of 20 millimoles per liter of blood was developed. The 2,3‐DPG level at four weeks of storage was proportional to DHA concentration in the 5 to 20 mM range. The osmotic fragility, red blood cell ATP levels, and plasma sodium, potassium, and hemoglobin during four weeks of storage in CPD‐adenine‐DHA were similar to those in blood stored in CPD‐adenine.     

Changes in adenosine triphosphate, 2,3 diphosphoglycerate, and P50 of dog blood following transfusion of autologous red cells pretreated with phosphoenolpyruvate in vitro

Red cells treated with phosphoenolpyruvate (PEP) in vitro were reinfused into the donor dogs and were monitored for changes in adenosine triphosphate (ATP), 2, 3 diphosphoglycerate (2,3 DPG), and P50. ATP and 2,3 DPG concentrations increased to 116 and 143 percent of control, respectively, when these cells were incubated with 60 mM PEP for 90 minutes at 37 degrees C. The oxygen dissociation curve shifted to the right, and P50 increased from 24.5 to 30.6 torr as a result of the PEP treatment. When one-half of the circulating red cell volume was treated with PEP and reinfused into the animal, the red cell 2,3 DPG increased to 120 percent of pretransfusion values. The 2,3 DPG level remained elevated during the following day and returned to near pretransfusion levels on the third day. The P50 of the circulating blood paralleled the variations in the red cell 2,3 DPG level, and the capacity for oxygen delivery was calculated to be raised by 13 to 38 percent for a period of 24 hours. In contrast, elevated red cell ATP returned to control values immediately after transfusion. In vivo viability, i.e., 24-hour survival and one-half disappearance time, of the cells pretreated with PEP were determined by a single-isotope technique using 51Cr. The results showed that PEP treatment did not injure the red cells. In addition, there was neither acute toxicity nor a deleterious hemodynamic effect when large amounts of PEP were administered intravenously. These results suggest that PEP could be used clinically to improve the capacity of the circulating red cells to deliver oxygen to the tissues.     

Xanthone additives for blood storage that maintain its potential for oxygen delivery. I. 2-Hydroxyethoxy- and 2-ethoxy-6-(5-tetrazoyl) xanthones in citrate-phosphate-dextrose-adenine (CPDA-1) blood

Two xanthones, 2-hydroxyethoxy-6-(5-tetrazoyl) (BW A440C) and 2-ethoxy- 6-(5-tetraozyl) (BW A827C), are members of a chemical series tested in vitro as potential additives to citrate-phosphate-dextrose-adenine (CPDA-1) medium for blood storage. P50 was maintained in the presence of these compounds during 42 days' storage by a partial maintenance of 2,3 diphosphoglycerate (2,3 DPG) and by a direct effect on hemoglobin previously reported for BW A827C. Red cell 2,3 DPG levels for BW A440C (n = 5), BW A827C (n = 5), and control (n = 6), respectively, were 3.38 +/− 0.47, 3.44 +/− 0.25, and 1.20 +/− 0.10 mM +/− SEM on day 7; 1.16 +/− 0.13, 1.52 +/− 0.37, and 0.16 +/− 0.02 mM on day 21; and 0.67 +/− 0.09, 0.61 +/− 0.08, and 0.06 +/− 0.006 mM on day 42. Red cell adenine triphosphate levels at the same time intervals were 1.84 +/− 0.09, 1.46 +/− 0.18, and 2.11 +/− 0.04 mM; 2.10 +/− 0.05, 2.07 +/− 0.17, and 2.13 +/− 0.05 mM; and 1.42 +/− 0.13, 1.37 +/− 0.13, and 1.38 +/− 0.06 mM, respectively. The degree of hemolysis was less with the addition of the compounds, and the methemoglobin formation, plasma Na+ and K+, and lactate production were unaffected by the compounds.     

Blood preservation 33. Phosphate enhancement of ribose maintenance of 2,3-DPG and ATP

Blood storage in CPD-adenine supplemented with 25 mM inosine and 10 mM phosphate gave 2,3-DPG levels as high as 140 per cent of normal for six weeks of blood storage at 4 C. Lower but normal 2,3-DPG levels were maintained throughout six weeks with inosine or inosine plus ribose. Ribose alone provided marginally increased DPG maintenance over the control, but ribose with phosphate maintained 2,3-DPG levels above 70 per cent of normal for five weeks of storage and two weeks longer than the control preservative. ATP levels were maintained at normal or above for six weeks with phosphate plus ribose or inosine. 2,3-DPG maintenance has previously been shown to be impaired by phosphate, unless inosine is also present. The ribose and inosine effects on 2,3-DPG maintenance are not additive. Phosphate also has an enhancement effect on ATP maintenance in the presence of either ribose or inosine.     

An improved red blood cell additive solution maintains 2,3‐diphosphoglycerate and adenosine triphosphate levels by an enhancing effect on phosphofructokinase activity during cold storage

BACKGROUND: Current additive solutions (ASs) for red blood cells (RBCs) do not maintain constant 2,3‐diphosphoglycerate (DPG) and adenosine triphosphate (ATP) levels during cold storage. We have previously shown that with a new AS called phosphate‐adenine‐glucose‐guanosine‐gluconate‐mannitol (PAGGGM), both 2,3‐DPG and ATP could be maintained throughout storage for 35 days. STUDY DESIGN AND METHODS: In this study, the mechanism underlying the effect of PAGGGM on RBC storage was studied in more detail. By using double‐erythrocytapheresis units (leukoreduced), a direct comparison could be made between the current AS saline‐adenine‐glucose‐mannitol (SAGM) and the experimental solution PAGGGM. During cold storage, several in vitro characteristics were analyzed. RESULTS: In agreement with our previous findings with single RBCs, PAGGGM maintained 2,3‐DPG and ATP levels for 35 days of cold storage. Furthermore, glucose consumption and lactate production were higher in PAGGGM units during the first 21 days of cold storage. Fructose‐1,6‐diphophate and dihydroxyacetone phosphate levels were also increased during the first 21 days of storage in PAGGGM units. CONCLUSION: These results indicate that it is likely that phosphofructokinase (PFK) activity is enhanced in PAGGGM units relative to SAGM units. After 21 days, PFK activity also decreases in PAGGGM units, but sufficient metabolic reserve in these units prevents depletion of 2,3‐DPG and ATP.     

A comparison of biochemical and functional alterations of rat and human erythrocytes stored in CPDA-1 for 29 days: implications for animal models of transfusion

Animal models of transfusion are employed in many research areas yet little is known about the storage-related changes occurring in the blood used in these studies. This study assessed storage-related changes in red blood cell (RBC) biochemistry, function and membrane deformability in rat and human packed RBCs when stored in CPDA-1 at 4 degrees C over a 4-week period. Human blood from five volunteers and five bags of rat RBC concentrates (five donor rats per bag) were collected and stored at 4 degrees C. RBC function was assessed by post-transfusion viability and the ability to regenerate adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) when treated with a rejuvenation solution. Membrane deformability was determined by a micropipette aspiration technique. ATP in rat RBCs declined more rapidly than human RBCs; after 1 week rat ATP fell to the same level as human cells after 4 weeks of storage (rat, 2.2 +/- 0.2 micromol g(-1) Hb; human, 2.5 +/- 0.3 micromol g(-1) Hb). Baseline DPG concentrations were similar in rat and human RBCs (16.2 +/- 2.3 micromol g(-1) Hb and 13.7 +/- 2.4 micromol g(-1) Hb) and declined very rapidly in both species. Human RBCs fully regenerated ATP and DPG when treated with a rejuvenation solution after 4 weeks of storage. Rat RBCs regenerated ATP but not DPG. Post-transfusion viability in rat cells was 79%, 26% and 5% after 1, 2 and 4 weeks of storage, respectively. In rats, decreased membrane deformability became significant (- 54%) after 7 days. Human RBC deformability decreased significantly by 34% after 4 weeks of storage. The rejuvenation solution restored RBC deformability to control levels in both species. Our results indicate that rat RBCs stored for 1 week in CPDA-1 develop a storage lesion similar to that of human RBCs stored for 4 weeks and underscores significant species-specific differences in the structure and metabolism of these cells.     

Synthesis of ITP and ATP and regeneration of 2,3-DPG in human erythrocytes incubated with adenosine, pyruvate and inorganic phosphate

In the stored, 2,3-DPG depleted human erythrocytes incubated for four hours in a medium containing adenosine (10 mM), pyruvate (10 mM), and inorganic phosphate (50 mM) the regeneration of 2,3-DPG reached the value of five times higher than the physiological concentration and the ATP synthesis exceeded four times the physiological level. It has been also found that these erythrocytes are able to synthesize hypoxanthine nucleotides, namely IMP to 58 and ITP to 61 mumoles per 100 ml of erythrocytes.     

Rejuvenation capacity of red blood cells in additive solutions over long-term storage

BACKGROUND: Red blood cells (RBCs) are Food and Drug Administration (FDA)‐approved for 42‐day storage with the use of additive solutions (ASs). However, adenosine triphosphate (ATP) and 2,3‐diphosphoglycerate (2,3‐DPG) levels in the RBCs decline over this time. These constituents may be restored by treatment with rejuvenation (REJ) solutions. This study was done to assess the response capability of RBCs from 30 to 120 days of storage in three FDA‐licensed RBC storage solutions after incubation with a rejuvenating solution of pyruvate, inosine, phosphate, and adenine. STUDY DESIGN AND METHODS: Three units each of RBCs in approved AS (AS‐1 [Adsol, Fenwal, Inc.], AS‐3 [Nutricel, Medsep Corp.], and AS‐5 [Optisol, Terumo Corp.]) were stored under standard conditions at 1 to 6°C for up to 120 days. Aliquots (4 mL) on Days 30, 42, 60, 80, 100, and 120 (±2 days) were REJ by incubating with Rejuvesol (Encyte Corp.). Control untreated and REJ aliquots were extracted using perchloric acid and stored at ?80°C until assayed for 2,3‐DPG and ATP. RESULTS: RBCs responded to REJ by increasing DPG and ATP contents. The response declined linearly at 0.070 ± 0.008 µmol DPG/g hemoglobin (Hb)/day and 0.035 ± 0.004 µmol ATP/g Hb/day with no differences between ASs. CONCLUSION: We conclude that Rejuvesol is able to restore ATP and 2,3‐DPG levels in RBCs stored up to 120 days in AS. The response diminishes as storage time increases. This rejuvenation (REJ) capability does not seem useful for routine assessment of RBC anabolic capacity in research programs, but may be useful to the investigator when studying unique and novel treatment methods.     

Characterization of biochemical changes occurring during storage of red cells. Comparative studies with CPD and CPDA-1 anticoagulant- preservative solutions

Citrate-phosphate-dextrose-adenine (CPDA-1), containing 0.25 mM adenine (final concentration) and 25 percent more glucose than citrate-phosphate-dextrose (CPD), has extended the allowable storage time for red cells to 35 days. Studies were conducted to understand better the characteristics of stored CPDA-1 red cells in relation to the properties of stored CPD red cells. Units with hematocrits near 80 percent showed the following: First, adenosine triphosphate (ATP) and total adenine nucleotide levels of red cells stored with CPDA-1 remained essentially constant during the first 3 weeks of storage after which the levels decreased; with red cells stored with CPD, ATP, and adenine nucleotide, levels were decreased even after 1 week of storage. Second, the pattern of the fall in 2,3-diphosphoglycerate was similar in red cells stored with CPD and CPDA-1. Third, changes in plasma and red cell levels of sodium and potassium, and in plasma ammonia levels, were comparable in CPD and CPDA-1 units; changes in cation levels were most pronounced during the initial 2 weeks of storage. Fourth, hemolysis was much greater in units stored in CPDA-1 for 35 days than in units stored in CPD for 21 days. Fifth, residual glucose concentrations were adequate in units drawn in CPDA-1 and stored for 35 days. We conclude that the changes in the biochemical characteristics of units of red cells stored with CPD and CPDA-1 are similar in most instances with the notable exception of the better maintenance of adenosine triphosphate levels in red cells stored with CPDA-1.     

Hemoglobin Function and 2,3‐DPG Levels of Blood Stored at 4 C in ACD and CPD pH Effect

Normal hemoglobin function depends on adequate erythrocyte levels of 2,3‐diphosphoglycerate (2,3‐DPG), a compound which is poorly maintained in acid‐citrate‐dextrose (ACD). Since 2,3‐DPG is better maintained in citrate‐phosphate‐dextrose (CPD) and this preservative has a higher pH (5.5) than ACD (pH = 5.0), these preservatives were prepared at each pH and studied. The CPD preservatives (pH 5.0, 5.5) had similar amounts of phosphate so the differences between them, obtained by altering the buffer ratio, should relate to pH. The ACD solutions (pH 5.0, 5.5) contained no phosphate. Hemoglobin function, expressed as P₅₀ (the Po₂ at 50 per cent oxygenation, an inverse but direct measure of oxygen affinity), and 2,3‐DPG were better maintained in ACD and CPD of pH 5.5. The lower pH (5.0) preservatives, whether ACD or CPD, showed rapidly declining hemoglobin function and 2,3‐DPG levels. The values at the higher pH remained close to normal for two weeks and above those of the lower pH preservatives for most of the four‐week storage period.     

Development of an optimized additive solution containing ascorbate-2- phosphate for the preservation of red cells with retention of 2,3 diphosphoglycerate

An additive solution containing adenine, ascorbate-2-phosphate, sodium phosphate, dextrose, and saline was developed for packed red cell preservation. The combination of all components was simultaneously optimized so that the resulting solution produced the maximum retention of both red cell adenosine triphosphate (ATP) and 2,3 diphosphoglycerate (2,3 DPG) concentrations. Fourteen nutrient combinations were tested; each combination was evaluated for 42 days of storage using cells from three donors. The nutrient combinations were chosen with the aid of a computerized experimental design process. Results of the experiments were modeled by regression analysis, and the model was optimized to produce the "best" formulation for simultaneous maintenance of ATP and 2,3 DPG. The resulting mathematically optimal formulation was tested in the laboratory using 10 units of red cells. With this solution, it was possible to store red cells for 42 days with retention of 45 to 55 percent of the initial ATP and 85 to 150 percent of the initial 2,3 DPG. Red cell lysis was low (0.8 percent), and most of the cells were biconcave discs (by scanning electron microscopy) at the end of storage. The studies were carried out in an efficient manner by using computer-optimized experimental design techniques coupled with multiple regression modeling and subsequent computer optimization of the models. This experimental approach has potential application to many current blood banking procedures. This additive solution should maintain viable red cells for 42 days. In addition, the solution will maintain red cell 2,3 DPG throughout storage.     

Blood preservation XXVII. Fructose and mannose maintain ATP and 2,3-DPG

Mannose and fructose as well as glucose have been shown to be effective for maintaining ATP and thus viability of stored red blood cells. Normal 2,3-DPG levels are desirable in stored red blood cells to provide the needed oxygen transport upon transfusion. ATP levels in sotred concentrated red blood cells in the new preservative, CPD- adenine (citrate-phosphate-dextrose-adenine) become critically low in the 5th week. In this study two hexoses and two pentoses are compared with dextrose in their ability to maintain ATP and 2,3-DPG. ATP levels were best maintained by fructose, then dextrose and mannose. ATP levels had fallen to critically low levels by four weeks with ribose and xylose. Red blood cell 2,3-DPG concentrations were also maintained by hexoses, with mannose being best, dextrose and fructose being similar. When ribose was used in addition to dextrose in CPD-adenine, ATP maintenance was improved and under the same conditions xylose improved 2,3-DPG maintenance. Fructose and mannose may be as useful as dextrose in citrate-phosphate preservatives for maintaining ATP and 2,3-DPG levels. Also, ribose and xylose may help the maintenance of ATP and 2,3- DPG, respectively, in CPD-adenine.     

In vitro evaluation of platelets stored in CDP-adenine formulations

Little information is available about the effect of adenine and added glucose on stored platelets. Two new formulations, CPDA-2 and CPDA-3, contain 34 mg adenine per 63 ml preservative and extra glucose (1.75 and 2.0 times the glucose in standard CPD). We have studied the in vitro integrity of platelet concentrates stored in CPD, CPDA-1, CPDA-2, and CPDA-3 at 22 C for 72 hours. Morphology score, pH, platelet size, population distribution parameters, and electron microscopic ultrastructure did not show any adverse effects which could be ascribed to the presence of adenine or extra glucose or both. No differences in platelet adenosine triphosphate (ATP) concentration or plasma glucose utilization during storage were found between CPD and CPDA-1 platelets. The results suggest that adenine and added glucose in these preservatives are not detrimental to platelets in vitro by the measures employed.