In vivo regeneration of red cell 2, 3-diphosphoglycerate following transfusion of DPG-depleted AS-1, AS-3 and CPDA-1 red cells

Regeneration of 2,3-diphosphoglycerate (DPG) was determined following transfusion of DPG-depleted group O red cells into group A recipients. Blood from five donors was stored in the adenine-containing solutions CPDA-1, AS-1 or AS-3 for 35 d at 4 degrees C. Post-transfusion red cell DPG and ATP were measured in separated group O red cells over a 7 d period. The studies confirmed rapid in vivo DPG regeneration with greater than or equal to 50% of the maximum level being achieved within 7 h. An average of 95% of the recipients' pre-transfusion DPG level was achieved by 72 h and by 7 d mean (+/- SEM) DPG levels relative to recipient's pre-transfusion DPG averaged 84% (+/- 13%), 92% (+/- 17%) and 84% (+/- 21%) for CPDA-1, AS-1 and AS-3 red cells, respectively. Results were comparable to those previously reported for blood stored in ACD for 15-20 d (Valeri & Hirsch, 1969; Beutler & Wood, 1969). The immediate regeneration rate, V, closely approximated first order regeneration kinetics with AS-3 red cells exhibiting double the rate of CPDA-1 red cells (P less than 0.001). AS-1 red cells exhibited an intermediate rate of regeneration which was not significantly different compared to either CPDA-1 or AS-3 (P greater than 0.05). V exhibited a significant (P less than 0.05) positive correlation with ATP levels 5-7 h post-infusion. ATP regeneration of the infused cells was rapid with a mean increase of 1.2 mumol/g Hb above post-storage levels being achieved 1 h following transfusion.     

Studies in red blood cell preservation. 3. A phosphate-ammonium-adenine additive solution

The maintenance of adenosine triphosphate (ATP) in red blood cells (RBC) during storage is largely dependent on the integrity of glycolytic metabolism in the Embden-Meyerhof pathway. Meryman et al. [Transfusion 1986; 26:500-505] hypothesized that a solution that increased the surface tension of the corpuscles through hypotonic swelling might retard the development of echinocytosis and membrane loss by the shedding of exocytic vesicles. Unexpectedly, maintenance of good ATP levels and satisfactory RBC survivals were found for a long as 18 weeks. The purpose of our study was to test their observations and to explore the possible mechanisms. Equal parts of units of packed RBC were stored in the experimental preservative and, for comparison, in ADSOL. The most notable findings were ATP values at 4 weeks averaging 5.2 mumol/g Hb (130% of initial) and at 12 weeks 2.9 mumol/g Hb (73% of initial), whereas these values declined as expected in ADSOL. Mean RBC diameters and surface areas by morphometric analysis were not significantly different in the two preservatives indicating the absence of any hypotonic swelling. The morphology scores of the RBC were significantly better throughout (p less than 0.05) than in ADSOL. The shedding of exocytic hemoglobin-containing vesicles was essentially the same in both preservatives. Our data confirm the observation that ATP levels are well maintained for at least 12 weeks, but do not show any evidence that hypotonic swelling was a part of the mechanism.     

24-hour 51Cr post-transfusion survival, 51Cr life span and haemolysis of red blood cells stored at 4 degrees C for 56 days in AS-3

BACKGROUND AND OBJECTIVES: Red blood cells (RBC) were collected either by a manual method using a 16-gauge needle or by an apheresis procedure using an 18-gauge needle, and were stored at 4 degrees C in a solution of CP2D (anticoagulant)/AS-3 (Nutricel) for 56 days. The purpose was to compare the outcome of the autotransfused red cells collected by both techniques. MATERIALS AND METHODS: Five healthy male volunteers were studied on two occasions. RESULTS: The autotransfusions of the manual and apheresed RBC resulted in a mean 24-h post-transfusion survival of 71%, a normal mean 51Cr RBC life span, a 2,3 DPG level that was less than 10% of normal, and 0.6% haemolysis. CONCLUSIONS: Whether collected manually or by apheresis, the outcomes were similar for RBC stored at 4 degrees C for 56 days in CP2D/AS-3.     

Temporal sequence of major biochemical events during Blood Bank storage of packed red blood cells

Background.

We used sensitive spectroscopic techniques to measure changes in Band 3 oligomeric state during storage of packed red blood cells (RBC); these changes were compared to metabolic changes, RBC morphology, cholesterol and membrane protein loss, phospholipid reorganisation of the RBC membrane, and peroxidation of membrane lipid. The aim of the study was to temporally sequence major biochemical events occurring during cold storage, in order to determine which changes may underlie the structural defects in stored RBC.

Materials and methods.

Fifteen RBC units were collected from normal volunteers and stored under standard blood bank conditions; both metabolic changes and lipid parameters were measured by multiple novel assays including a new mass spectrometric measurement of isoprostane (lipid peroxidation) and flow cytometric assessment of CD47 expression. Band 3 oligomeric state was assessed by time-resolved phosphorescence anisotropy, and RBC morphology by microscopy of glutaraldehyde-fixed RBC.

Results.

Extracellular pH decreased and extracellular potassium increased rapidly during cold storage. Band 3 on the RBC membrane aggregated into large oligomers early in the storage period and coincident with changes in RBC morphology. Membrane lipid changes, including loss of unesterified cholesterol, lipid peroxidation and expression of CD47, also changed early during the storage period. In contrast loss of acetylcholinesterase activity and haemolysis of RBC occurred late during storage.

Discussion.

Our results demonstrate that changes in the macromolecular organisation of membrane proteins on the RBC occur early in storage and suggest that lipid peroxidation and/or oxidative damage to the membrane are responsible for irreversible morphological changes and loss of function during red cell storage.     

Storage of ADSOL-preserved red cells at 2.5 and 5.5 degrees C: comparable retention of in vitro properties

Previous studies with CPDA-1 and Nutricel preserving solutions indicated that red cell properties were affected by small differences in storage temperature. The influence of a 3 degrees C differential on the preservation of the in vitro properties of ADSOL-preserved (AS-1) red blood cells was investigated in a paired study. AS-1 red blood cells were stored at 2.5 and 5.5 degrees C for 42 days. Extent of hemolysis, glucose consumption and pH levels were comparable at the two storage temperatures. Lactate levels were slightly, but significantly higher at 5.5 degrees C. ATP levels were slightly but significantly higher at 2.5 degrees C, only during the later part of the storage period. 2.3-DPG levels were slightly better retained at 2.5 degrees C after 7 days of storage. Holding units of whole blood for either 1 or 8 h at ambient temperature after phlebotomy prior to processing did not influence the types of temperature-dependent changes. The differences as a function of storage temperature were small and appear to be of no practical importance in connection with the storage of AS-1 red blood cells.     

Studies in Red Blood Cell Preservation 1. Effect of the Other Formed Elements

The purpose of this study was to determine if the removal of most of the leukocytes and platelets would affect the in vitro characteristics of stored red blood cells (RBC). Fresh RBC concentrates prepared by removing platelet-rich plasma and filtration through Imugard IG 500 filters were compared with unmanipulated units after storage for up to 56 days. The filtered units were significantly better after 56 days storage for supernatant K+ (p = 0.001), hemolysis (p = 0.05), total vesicle membrane protein shed (p = 0.03), and RBC morphology score (p = 0.04). These differences occurred even though ATP levels were well maintained in both groups. The measurements that did not differ significantly were pH, hematocrit, ATP, 2,3-DPG, glucose and supernatant Na+. It is suggested that enzymes, leukotrienes, catecholamines and eicosanoids released by degenerating leukocytes and platelets may be inimical to RBC. Some may act as agonists on alpha-adrenergic and cholinergic muscarinic receptors present on RBC membranes.     

Studies in Red Blood Cell Preservation: 5. Determining the Limiting Concentrations of NH4Cl and Na2HPO4 Needed to Maintain Red Blood Cell ATP during Storage

The purpose of the present study was to define the lowest concentrations of ammonium (NH4+) and phosphate (Pi) in an experimental additive solution (EAS) that would support suitable red blood cell (RBC) ATP levels and other in vitro characteristics for at least 84 days. It was determined that ATP maintenance was dependent upon both NH4+ and Pi concentrations. RBCs stored for 84 days in additive solutions containing 10 mM NH4+ and 0, 15, 25 and 40 mM Pi had ATP values averaging 1.87, 2.49, 2.70 and 2.65 mumol/g Hb, respectively. The shedding of exocytic hemoglobin-containing vesicles and percent hemolysis were significantly (p less than 0.001) elevated in the preservative containing 40 mM Pi. These data suggest that an EAS containing 10 mM NH4+ and 15 mM Pi would be optimal for storing RBCs up to 84 days. The extended storage would be particularly advantageous for autologous transfusion programs.     

Improved red blood cell preservation correlates with decreased loss of bands 3, 4.1, acetylcholinestrase, and lipids in microvesicles

In earlier studies we have shown that a final concentration of 0.69% glycerol in blood mixed with an experimental additive solution, EAS 25, improves the in vitro quality and in vivo survival of red blood cells (RBCs). The objective of this study was to determine if the better preservation of RBCs in EAS 25 is correlated with the improved maintenance of membrane lipids and proteins and decreased vesiculation. Split units of RBCs were stored in Adsol or EAS 25 (mmol/L: adenine 2/2, dextrose 122/110, mannitol 42/55, glycerol 0/150, NaCl 154/50). After 12 weeks storage, RBC and microvesicle membranes were analyzed for cholesterol, phospholipid, diphenyl hexatriene fluorescence anisotropy, and acetylcholinesterase (AchE) activity. Bands 3 and 4.1 were identified in the microvesicle membranes by immunoblotting. The RBC membrane cholesterol, phospholipids, and AchE remained higher in EAS 25 than in Adsol (P < .001). Vesicle membrane lipids and AchE in EAS 25 were significantly less than in Adsol (P < .001). The fluidity of stored cells in both the solutions was greater than the prestorage samples. Immunoblotting analyses showed that bands 3 and 4.1 were greatly reduced in the microvesicle membranes shed by the RBCs stored in EAS 25 compared with those formed in Adsol.     

The Role of Mean Corpuscular Haemoglobin Concentration in Limiting the Storage Life of Human Blood

Human erythrocytes stored for more than 21 days in citrate phosphate dextrose with adenine (CPDA-1) at +4 degrees C had a decreased cell volume, an increased cell density, an elevated mean corpuscular haemoglobin concentration (MCHC) and hence an elevated internal viscosity. These changes are normally masked by the reversible cell swelling which accompanies storage in CPDA-1. Incubation of stored cells for 24 h at 37 degrees C in fresh autologous plasma mimics the effects of reinfusion and causes the artificially swollen cells to shrink to their true volume. Thus, incubated cells which had been stored in CPDA-1 for more than 21 days exhibited a decreased filterability through Nuclepore membranes in vitro, which is apparently correlated with a decreased survival time in vivo.     

Studies in Red Blood Cell Preservation

The purpose of this study was to examine whether vesiculation of RBC plays a significant role in their rejuvenation. Outdated units of Adsol blood, were divided into two aliquots and incubated with equal volumes of a solution of 100 mM pyruvate and inosine, 103 mM phosphate and 5 mM adenine (PIPA) or 0.9% saline. Following 1 h incubation, vesicles were isolated from the supernatants and quantitated for hemoglobin content. Restoration of RBC ATP, 2,3-DPG, morphology, and osmotic fragility after rejuvenation was satisfactory. The postrejuvenation mean corpuscular volumes (88.2 +/- 6.9 fl) were significantly lower (p less than 0.001) than the prerejuvenation (94.6 +/- 6.8 fl) and control (104.0 +/- 7.3 fl) volumes. The hemoglobin shed in vesicles during rejuvenation was significantly greater than in the saline controls (0.44 +/- 0.31 vs. 0.18 +/- 0.10 mg/dl RBCs; p = 0.026). These data suggest that the decreased MCV following rejuvenation is in part due to membrane loss in exocytic vesiculation.     

The storage of hard-packed red blood cells in citrate-phosphate-dextrose (CPD) and CPD-adenine (CPDA-1).

The preservation of red cells "hard packed" to a hematocrit of over 80% from blood collected in citrate-phosphate-dextrose (CPD) or CPD-adenine (CPDA-1) has been investigated. After 21 days of storage, cells that had been collected in CPD solution had consumed most or all of the available glucose and manifested markedly impaired viability after reinfusion into the normal donor. In contrast, red cells prepared from blood collected in CPDA-1, a medium containing supplementary adenine and an increased amount of glucose, maintained higher glucose and adenosine triphosphate levels and, in most instances, manifested satisfactory posttransfusion viability. We emphasize that in addition to providing longer shelf life of stored blood, CPDA-1 provides a better hard-packed red cell concentrate for transfusion at 21 days.     

Contribution of the spleen to the lipid metabolism in plasma and red cells. On hereditary spherocytosis and hereditary high red cell membrane phosphatidylcholine hemolytic anemia

Clinical and experimental studies on hereditary spherocytosis (HS) and high red cell membrane phosphatidylcholine hemolytic anemia (HPCHA) were performed in relation to lipid metabolism in plasma and in red cells of these patients. In HS, red cell (RBC) membrane lipids (free cholesterol (FC) and phospholipids (PL) such as phosphatidylethanolamine, phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC] were markedly decreased in unsplenectomized HS. Plasma lipids (total cholesterol, FC, HDL-cholesterol, PL) were also decreased in these patients. After splenectomy, substantial normalization of plasma and RBC lipids were observed. Concerning lipid kinetics, the extents of 14C-PC synthesis in RBC from 14C-LPC in medium and of 14C-PC exchange between RBC and the medium were almost identical to those in normal control in the in vitro incubation conditions. These observations indicate that the decreased RBC lipids may be induced by the shortage of lipid materials in plasma in the presence of the spleen. In unsplenectomized HPCHA, plasma lipids were also decreased same as in HS. In contrast, membrane lipids (FC and PC) were markedly increased. Even after splenectomy, increased PC contents were rather enhanced in their membranes concomitant to developing of hemolytic anemia, although plasma lipids were almost normalized. Thus RBC membrane lipids in HPCHA appeared not to be affected by plasma lipids. In experimental studies on HPCHA, 14C-PC synthesis from 14C-LPC and 14C-PC uptake in these RBC were increased, in spite that large amounts of PC were already accumulated in these RBC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythrocyte deformability in diabetes and erythrocyte membrane lipid composition

Erythrocyte deformability was assessed in 40 diabetic patients, 24 insulin-dependent (IDD) and 16 non-insulin-dependent (NIDD), by measuring the initial filtration flow rate of whole blood, isolated red blood cells (RBC), and isolated RBC membranes with the Hanss hemorheometer, and its relationship to the plasma and ghost membrane lipid composition was investigated. RBC deformability was significantly reduced, whereas the deformability of the isolated RBC membranes did not differ significantly from the controls. In the plasma, the triglycerides were high, the high-density lipoprotein (HDL) cholesterol was reduced, and the ratio of total cholesterol over HDL cholesterol was high as compared with the controls. The RBC lipid composition expressed in mumol lipids/10(10) RBC showed significantly lower levels of free cholesterol, sphingomyelines, and phosphatidylcholine, which are the lipids principally located on the outer layer of the RBC membranes. These data suggest that in both IDD and NIDD patients, there may be a relation between these modifications in the RBC lipid composition and rheological impairment of the RBC.     

Erythrocyte Membrane Vesiculation and Changes in Membrane Composition during Storage in Citrate-Phosphate-Dextrose-Adenine-1

Serial studies were made of the membranes of the erythrocytes and the vesicles shed during storage of blood in polyvinyl chloride containers for 35 days in citrate-phosphate-dextrose-adenine anticoagulant. Special precautions were taken to eliminate artifacts created by contaminating leukocytes, platelets and red blood cell ghosts. A total of 15.6% of the cholesterol and 5.2% of the phospholipids of the membranes was lost with no gross change in the gel electrophoretic patterns. The quantity of vesicles found in the supernatant plasma increased during storage and their membranes were characterized by the absence of spectrin, ankyrin, and periodic acid Schiff bands 2 and 3. The ratio of lipids to protein in the vesicles increased as they accumulated perhaps reflecting a rearrangement of the erythrocyte membrane constituents during prolonged maintenance at 4 degrees C.     

Donor sex,age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity

Red blood cell (RBC) storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, the metabolic underpinnings of the capacity of RBC to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBC from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyse following oxidant stress. A subset of extreme hemolysers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBC from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBC stored in additive solution- 3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBC stored in additive solution-1. Merging metabolomics data with results from an independent genome-wide association study on the same cohort, we identified metabolic markers of hemolysis and glucose 6-phosphate dehydrogenasedeficiency, which were associated with extremes in oxidative hemolysis and dysregulation in nicotinamide adenine dinucleotide phosphate and glutathione- dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and glucose 6-phosphate dehydrogenase status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults.     

Spironolactone- and canrenone-induced changes in hepatic (Na+,K+)ATPase activity, surface membrane cholesterol and phospholipid, and fluorescence polarization in the rat

We studied changes in hepatic membrane (Na+,K+)ATPase activity and membrane lipids induced by canrenoate, the water-soluble congener of canrenone, the active metabolite of spironolactone. (Na+,K+)ATPase activity was decreased after canrenoate in a dose- and time-dependent manner. Decreased activity was demonstrated at the lowest dose (91% of control after 5 mumoles per 100 gm body weight per day X 3 days); the maximum dose (30 mumoles per 100 gm body weight per day X 3 days) resulted in activity 38% of untreated control values. A 20 mumoles per 100 gm body weight per day dose decreased enzyme activity to 89 and 55% of control after 24 and 72 hr, respectively. The nonionic detergent Triton WR-1339 partially reversed drug-induced inhibition, suggesting that the enzyme changes may be related to altered membrane lipids. Membrane cholesterol increased 17% after 3 days of 30 mumoles canrenoate per 100 gm body weight per day; phospholipids decreased by 12%. The cholesterol to phospholipid molar ratio increased from 0.419 to 0.555. Membrane fluidity, as measured by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene decreased after treatment with 20 mumoles canrenoate per 100 gm body weight per day for 3 days. These results describe in vivo and in vitro inhibition of hepatic (Na+,K+)ATPase activity. Increased membrane cholesterol with decreased phospholipid alters membrane fluidity and may be partially responsible for the change in (Na+,K+)ATPase activity.     

Platelet Storage Lesions in Second-Generation Containers: Correlation with in vivo Behavior with Storage up to 14 Days

The relationship between in vivo behavior and in vitro characteristics of 59 platelet concentrates (PC) stored for up to 14 days in a synthetic medium or in CPDA-1 plasma was systematically investigated. 25 paired studies (1 study was incomplete) were performed comparing platelets suspended either in the synthetic medium or CPDA-1 plasma with 5 days (n = 5); 7 days (n = 10); 10 days (n = 5); and 14 days (n = 5) of storage. In addition, 10 control studies were performed with freshly prepared PC (6-24 h) in CPDA-1 plasma. Both percent recovery and survival estimations showed decreases with increasing storage duration, irrespective of storage medium used. In both media, with prolonged storage, the platelet survival curves not only became shorter, but also increasingly exponential, suggesting that in vitro storage caused progressive damage to the platelets present in circulation. Survival curves of platelets suspended in synthetic medium remained more linear, indicative of less random damage during storage. Mean population lifespan (MPL) of the stored PC was determined by the area below the survival curve divided by the mean percent recovery for the fresh PC, which was 55%. MPL decreased from 4.5 days (fresh PC) to 0.4 days after 14 days of storage in plasma, with a 50% reduction (t1/2) estimated at 7.2 days of storage. MPL t1/2 for PC stored in the synthetic medium was estimated to be 8.8 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gamma-ray-irradiated red blood cells stored in mannitol-adenine-phosphate medium: rheological evaluation and susceptibility to oxidative stress

BACKGROUND AND OBJECTIVES: To evaluate the rheological properties and the oxidative susceptibility of gamma-ray-irradiated red blood cells (RBCs). MATERIALS AND METHODS: RBCs in mannitol-adenine-phosphate (MAP) medium were irradiated with 35 Gy and stored at 4 degrees C for 4 weeks. The deformability of the RBCs was examined under shear flow in relation to the morphological and biochemical changes. The RBCs were further exposed to 1 mM FeSO(4) and 5 mM ascorbate to examine the oxidative susceptibility. RESULTS: The RBC deformability was decreased during storage, and the impairment was further enhanced by the irradiation, which promoted cell shrinkage and intracellular hemoglobin condensation accompanying potassium loss. Lipid peroxidation and protein aggregation of the RBC membrane as well as echinocytosis were not enhanced by the irradiation. The exposure to free iron did not stimulate the oxidation of the irradiated RBC membrane. CONCLUSION: The decreased deformability of gamma-ray-irradiated RBCs in MAP medium was mainly induced by dehydration due to potassium loss, and the membrane lipids and proteins were stably preserved against oxidative stress.     

The Erythrocyte Membrane Disturbances in Protein-Energy Malnutrition: Nature and Mechanisms

In protein-energy malnutrition (PEM), as observed in Kivu, the RBC have an increased ratio of surface area to volume which is demonstrated by the presence of target cells on light microscopy and cup cells with scanning electron microscopy. The osmotic fragility is decreased. These abnormalities can be attributed to the accumulation of cholesterol and phosphatidylcholine (PC) in the RBC membrane. The molar ratio of cholesterol to phospholipids is moderately increased. Several findings suggest that the cholesterol and PC build-up results from disturbed exchanges in these lipids between the RBC and the plasma lipoproteins. Firstly, the osmotic fragility of a patient's RBC gradually becomes normal when the cells are transfused into a healthy recipient. Secondly, the cholesterol flux between the RBC and the plasma LDL seems to be low. Thirdly, the increase in RBC PC cannot be explained by a diminished fatty acids transport between the deep RBC PC pool and the RBC phosphatidylethanolamine (PE) pool. Finally complex disturbances of the plasma lipoproteins are obvious. It is improbable that the cholesterol and PC build-up accounts for the premature RBC destruction which has been described in Kivu PEM. However, the observation of an increased fatty acid turnover in RBC PC and PE, as well as other data previously obtained in Kivu PEM, lead to the conclusion that membrane peroxidation may be a major cause of the shortened erythrocyte life-span in this syndrome.     

Red blood cell storage in SAGM and AS3: a comparison through the membrane two-dimensional electrophoresis proteome

BACKGROUND: SAGM is currently the standard additive solution used in Europe, while AS-3 is the third additive solution that has been licensed in the USA, and is also the one used in part of Canada. Although AS-3 is based on a saline-adenine-glucose solution, it also contains citrate and phosphate. Storage of red blood cell concentrates in CPD-SAGM is known to lead to the accumulation of a wide series of storage lesions, including membrane protein fragmentation and vesiculation, as we could previously determine through 2-dimensional gel electrophoresis. MATERIALS AND METHODS.: Through 2D-SDS-IEF-polyacrilamide gel electrophoresis we performed a time course analysis (day 0, 21 and 42 of storage) of red blood cell membranes from leukocyte-filtered concentrates either stored in CPD-SAGM or CP2D-AS-3. RESULTS AND DISCUSSION.: From the present study it emerges that the membrane protein profile of red blood cells stored in presence of AS-3 appears to be slightly different from (better than) previous reports on SAGM-stored counterparts. However, the increase of total membrane spot number due to the presence of fragments at day 21 and the significant decrease at day 42 are suggestive of a universal phenomenon which is not efficiently tackled by either of the two additive solutions investigated in the present study. CONCLUSION: To further delve into the storage lesion issue for RBCs stored in AS-3, it would be interesting in the future to assay metabolic changes over storage progression as well.