Studies of Factors Regulating the Ageing of Human Erythrocytes

Erythrocytes from human blood stored at 5 degrees C in anticoagulant-preservative solutions are considered, after a period from 21 to 24 days, unsuitable for transfusion. The nonviability of the cells appears due to metabolic impairment accompanied by a change of the biophysical properties of the cell but surprisingly not to a decrease of sialic acid of the membrane. The absence of proteolysis can be attributed to calcium chelation and to the stable levels of plasma protease inhibitors throughout the storage period. In fact, when erythrocytes are incubated for several days at 5 degrees C in protein-free media, release of sialopeptides from the membrane increases slowly and steadily, particularly if the medium is without glucose and adenine but contains divalent cations. Moreover, this phenomenon is strikingly amplified when incubation in protein-free media is carried out at 37 degrees C and once the ATP level has fallen below 5% of its initial value.     

Studies on the Metabolism of Adenosine and Adenine in Stored and Fresh Human Erythrocytes

The metabolism of adenosine-8-C¹⁴, at relatively high and low concentrations, was studied in human erythrocytes freshly obtained and after 6 and 9weeks of storage in ACD. At high adenosine concentration (3.6 µM/ml. cells),considerable conversion to ATP was found in fresh and stored cells, suggestingthat direct phosphorylation of adenosine occurred, a reaction that is minimalat low (0.36 µM/ml. cells) adenosine concentration because of extensive rapiddeamination. Incorporation of the label via hypoxanthine or adenine is unlikely,since at high adenosine concentration no dilution of ATP labeling in thepresence of unlabeled inosine (hypoxanthine) was found, nor was free labeledadenine detected in erythrocyte extracts.

A study of the metabolism of adenine-8-C¹⁴ in fresh and stored erythrocytessuggests that the presence of a suitable nucleoside (e.g., inosine) is requiredfor efficient utilization of adenine-8-C¹⁴ for ATP formation in the erythrocytesof blood stored for prolonged periods.

Submitted on June 24, 1965 Accepted on August 23, 1965     

Biochemical Studies on McLeod Phenotype Erythrocytes

Abstract. Red cells of the McLeod phenotype in the Kell blood group system have an acanthocytic morphology. The membrane protein composition analyzed on sodium dodecylwlfate-polyacrylamide gel electrophoresis, the ATP level and the activities of a large number of intracellular enzymes appear to be normal. Membranes prepared from McLeod red cells incubated with γAT[³²P] and MgCl₂ incorporated twice as much radioactivity into spectrin and also showed a slight elevation of phosphorylation in band 3 protein when compared to membranes from normal cells. Intact normal red cells incubated with carrier-free [³²P] incorporated radioactivity into several proteins. with most incorporation in spectrin and band 3 protein. In comparison, McLeod cells incorporated three times more radioactivity into spectrin and band 3 protein but increased phosphorylation also occurred in other, but not all, membrane proteins. Intact McLeod red cells also showed increased phosphorylation of membrane phospholipids, but they incorporated [³²P] into intracellular nucleotide phosphates in a normal manner.     

Metabolism of Guanosine in Human Erythrocytes

The metabolism of guanosine in human erythrocytes has been studied in two different experimental systems--direct incubation and dialysis incubation--the latter allowing continuous addition and removal of substances. Intra- and extracellular purine compounds were analyzed using high-performance liquid chromatography (HPLC). At 37 degrees C, a normal pH (7.4) and a favorably high concentration of inorganic phosphate, guanine nucleotides were synthesized at a substance rate of about 0.17 mmol . h-1 (calculated per liter erythrocytes) when guanosine was kept at a concentration of 25 mumol . l-1. At a higher guanosine concentration the rate of synthesis increased only moderately. Erythrocytes loaded with guanylates lost these nucleotides at a rate of 0.023 mmol . h-1 at a normal phosphate concentration and somewhat slower at a higher phosphate concentration. The metabolism kept the guanylates in an equilibrium that was similar to the equilibrium between the adenylates.     

Studies on Glycoproteins and Glycopeptides from Tn-Polyagglutinable Erythrocytes

Abstract. Direct evidence for the theory that Tn-polyagglutinable erythrocytes have a deficiency of alkali-labile sialic acid and galactose is gained by analysing the carbohydrate moiety of the tryptic glycopeptides with spectrophotometric methods and gas-liquid chromatography. Alkaline borohydride treatment of these glycopeptides specifically releases N -acetylgalactosaminitol. In addition it is shown that mainly the third and the first membrane glycoprotein are affected by the defect.     

Autoradiography of Diffusible Compounds in Human Nonnucleated Erythrocytes: Studies with Tritiated Glucose and Adenine

A simple method for autoradiography of diffusible substances in erythrocytes is described. It is based on retarding diffusion from the red corpusclesduring fixation and processing by using the bifunctional aldehyde, glutaraldehyde; rapid drying; and applying the nuclear emulsion as a dried, preformedfilm.

The technique was used for studying the changes during storage in theability of red blood cells to utilize glucose. The incorporation of glucose-6-³Hby samples of blood stored for different periods of time was followed by thenumber of silver grains present in the red corpuscles. It was observed thatthe percentage of erythrocytes with fewer than four grains increased progressively during the storage of ACD blood. The incorporation of glucose asobserved by the silver grain pattern was reversible toward that of freshly collected red corpuscles following incubation of the stored samples withadenosine.

Similar studies were performed using adenine-8-³H as the label in bloodstored for six weeks in ACD adenine solution. Incubation of the blood samplescontaining 0.5 mM. adenine for one hour at 37°C resulted in autoradiographicpatterns equivalent in grain counts to those given by red blood cells storedwith the radiolabeled adenine for five weeks. The incubation of fresh sampleswith inosine was found to inhibit the incorporation of adenine, whereas inosineenhanced the number of grains in red blood cells which had "run down"during storage.

The autoradiographic technique presented should be of value for the in vitroevaluation of methods of blood preservation and for studying the metabolicheterogeneity of normal red corpuscles and other tissue cells. It should be useful for investigating hereditary and acquired abnormalities of metabolism.

     

Morphology of Stored, Rejuvenated Human Erythrocytes

Abstract. Whole blood was stored at 4°C for 42 days. At 7-day intervals, starting with day 21, aliquots were rejuvenated with a phosphate, inosine, glucose pyruvate, and adenine solution. Samples were taken for ATP assays and red cell morphology (by SEM) both before and after rejuvenation. Rejuvenation elevated all ATP levels in excess of 100% of the starting values and significantly increased the percent of cells in the discoid shape as measured by their morphological index, showing that rejuvenation not only raises the cell's metabolic level but causes partial reversion to fresh cell morphology.     

Machine Learning Method for Fatigue Strength Prediction of Nickel-Based Superalloy with Various Influencing Factors

The accurate prediction of fatigue performance is of great engineering significance for the safe and reliable service of components. However, due to the complexity of influencing factors on fatigue behavior and the incomplete understanding of the fatigue failure mechanism, it is difficult to correlate well the influence of various factors on fatigue performance. Machine learning could be used to deal with the association or influence of complex factors due to its good nonlinear approximation and multi-variable learning ability. In this paper, the gradient boosting regression tree model, the long short-term memory model and the polynomial regression model with ridge regularization in machine learning are used to predict the fatigue strength of a nickel-based superalloy GH4169 under different temperatures, stress ratios and fatigue life in the literature. By dividing different training and testing sets, the influence of the composition of data in the training set on the predictive ability of the machine learning method is investigated. The results indicate that the machine learning method shows great potential in the fatigue strength prediction through learning and training limited data, which could provide a new means for the prediction of fatigue performance incorporating complex influencing factors. However, the predicted results are closely related to the data in the training set. More abundant data in the training set is necessary to achieve a better predictive capability of the machine learning model. For example, it is hard to give good predictions for the anomalous data if the anomalous data are absent in the training set.     

Magnesium Metabolism of Human and Rabbit Erythrocytes

A modification of the magnesium ammonium phosphate precipitation methodfor the determination of serum magnesium was devised to determine themagnesium content of erythrocytes.

The concentration of magnesium in the red cells of healthy hospital personnel was 4.67 ± 0.92 mEq./L.

An increase in erythrocyte magnesium concentration was observed inpatients with reticulocytosis. Experimental production of reticulocytosis bythe administration of phenylhydrazine to rabbits confirmed these clinicalobservations.

No significant in vitro uptake of Mg²⁸ from the suspending medium occurred in mature human erythrocytes or in mature or immature erythrocytesfrom rabbits.

The relative tissue uptake of Mg²⁸ in the bone marrow was significantlyincreased in animals in whom anemia and marked reticulocytosis were produced by phenylhydrazine. Relative activity was decreased in the hearts,spleens, and kidneys of these animals.

Since there is no evidence for significant exchange of magnesium in immature or mature erythrocytes in the peripheral circulation, it is concludedthat the magnesium content of erythrocytes is increased in the bone marrowprior to their release into the peripheral circulation.

Submitted on April 16, 1962 Accepted on May 28, 1962     

Fumarase Activity of Human Leukocytes and Erythrocytes

1. The results of over 400 assays of fumarase activity in separated leukocytesand erythrocytes in a variety of hematologic and nonhematologic diseases arepresented.

2. The highest mean leukocyte fumarase values occurred in acute monocyticleukemia and acute granulocytic leukemia. Blast cells have greater fumaraseactivity than more mature forms of the same cell type.

3. Fumarase appears to be distributed ubiquitously among the various leukocytes. Cell populations which contained predominantly neutrophils, lymphocytes, monocytes, blasts, eosinophils, and basophils, all exhibited appreciableenzyme activity.

4. Assays of erythrocyte fumarase activity did not reveal a pattern with theexception of elevated values in macrocytic anemias and in some, but not all,cases of hemolytic anemia.

Submitted on August 22, 1960 Accepted on December 9, 1960     

Dependence of Purine Loss from Human Erythrocytes on External pH

S ummary . Human red cells were incubated at 37°C in physiological saline at pH 7.4 with 8-³H-adenine to label the adenine nucleotides. After washing, the cells were incubated in similar salines which were buffered to different values of pH. The loss of radioactivity from cells into medium was very slow below pH 7.4 and accelerated greatly with increasing pH up to about 7.8. At that pH the loss increased for at least 3 h when it represented more than 30% of the initial radioactivity of the cells. The loss could not be accounted for by haemolysis which remained minimal. The extracellular radioactivity was present mostly in hypoxanthine and some inosine. The loss of purine did not depend on oxygenation of the cells. These results, together with the high-affinity uptake system for adenosine, could provide a partial explanation of the turnover of adenine nucleotide purines of red cells which is many times faster than that of the cells themselves.     

The Effect of the Nitrite Ion on Intact Human Erythrocytes

The mode of action of the nitrite ion on intact human erythrocytes has beeninvestigated under varying experimental conditions. Nitrite appeared to actdirectly to cause methemoglobin formation and GSH depletion. Evidencewas presented to show that such depletion does not occur in the presence ofa normal and active pentose phosphate pathway, and to suggest that methemoglobin formation buffers the erythrocyte against nitrite-induced oxidationof GSH.

Both in the presence and absence of glucose, nitrite in high concentrationwas found to cause increased rates of oxidative destruction of hemoglobinwith Heinz body formation, and of loss of osmotic integrity. It was suggestedthat nitrite acts by "simple" oxidation of the -SH groups of globin and othererythrocytic components, and possibly by causing deamination of cellularproteins.

The results were discussed, with reference to the significance of cellularprotective mechanisms under evolutionary or environmental conditions whichinvolve intermittent exposure to high concentrations of the nitrite ion.

Submitted on February 6, 1962 Accepted on July 3, 1962     

The Effects of Cisplatin on Normal Human Erythrocytes in Vitro

Therapeutic administration of cisplatin is often followed by anaemia which may not be due entirely or even largely to myelosuppression but rather to drug-induced haemolysis. Similar experience with other cytotoxic agents prompted earlier investigations which yielded evidence of drug-induced changes in RBC shape in vitro. Current studies of cisplatin and bleomycin, which have been used together in clinical practice, revealed a significant alteration in the profile of RBC morphology; specifically a reduction in the number of ‘bowl’ shaped cells. However, this finding is in contrast to the increase in ‘bowls’ which was observed previously with other drugs. So it appears that direct erythrocytotoxicity cannot explain the occurrence of anaemia in patients who receive cisplatin. An alternative mechanism, similar to that of penicillin induced haemolysis, is suggested by immunoradiometric assays of surface IgG on red cells.     

Research on Mechanical Properties and Influencing Factors of Cement-Graded Crushed Stone Using Vertical Vibration Compaction

To study the mechanical properties of cement-graded crushed stone for use in the transition sections of intercity railways, the growth laws governing unconfined compressive strength, splitting strength and resilience modulus of cement-graded crushed stone and their influencing factors were studied by the vertical vibration compaction method (VVCM). The strength growth equations of cement-graded crushed stone are proposed, and strength prediction equations are established. The research shows the unconfined compressive strength, splitting strength and resilience modulus of cement-graded crushed stone with a strong interlocked skeleton density type (VGM-30) are significantly enhanced to 20, 20 and 17% higher, respectively, than those of standard cement-graded crushed stone. The growth law of mechanical properties of cement-graded crushed stone is similar, with the fastest growth occurring before 14 days, and the rate decreasing after 28 days. The strength growth tended to be stable after 90 days, increasing with the increase in curing time, compaction coefficient and cement dosage. The correlation coefficients (R²) of the strength growth prediction models were found to be 0.99, 0.97, and 0.99, respectively. These values can be used to accurately predict the strength growth curve. This paper verifies the superiority of VGM-30 gradation through laboratory tests, providing a reference for gradation selection in the construction of intercity railway transition sections.     

Ultrastructural Studies of Leucocytes which Form Rosettes with Homologous Erythrocytes in Human Auto Immune Haemolytic Anaemia

Leucocytes which form rosettes with homologous erythrocytes in auto-immune haemolytic anaemia, infectious mononucleosis and following the stimulation of lymphocyte cultures with PHA have been studied by electron microscopy. The studies showed that the predominant homologous rosette forming cell was lymphocytic and the evidence favoured the conclusion that they were non-immune in nature. This identification does not classify the lymphocyte as thymus or bone marrow derived. Just under half (44%) of the total lymphocytic RFC had perinuclear microfilaments and limited clearing of the plasma of ribosomes close to the cell membrane. These features are consistent with activated T lymphocytes. The remainder of the lymphocytic RFC had no features which allowed their identification as T or B cells. The form of contact between lymphocytic RFC and erythrocyte was mainly of a pointlike nature, with little deformation or invasion of the attached erythrocytes. Monocytes were the only other type of cell encountered with any frequency. Their prime distinguishing feature was the deformation of attached red cells which they caused.     

ICU机械通气患者发生2型心肌梗死的影响因素研究

背景心肌氧供需失衡是导致2型心肌梗死(T2MI)发生的主要因素,而机械通气是患者因机体缺氧而采取的治疗方式,故推测机械通气患者T2MI发生风险高,但目前临床针对该类患者的T2MI诊断存在一定难度。目的探讨ICU机械通气患者发生T2MI的影响因素,以期为临床识别T2MI高风险人群提供参考。方法选取2017—2019年上海健康医学院附属嘉定区中心医院ICU收治的危重症患者585例,经纳入与排除标准最终纳入287例机械通气患者,根据T2MI发生情况将其分为T2MI组130例和非T2MI组157例。比较两组的一般资料、疾病类型、氧供需失衡因素。采用多因素Logistic回归分析探讨ICU机械通气患者发生T2MI的影响因素。结果T2MI组患者糖尿病发生率、慢性心力衰竭发生率、急性生理学及慢性健康状况评分系统Ⅱ(APACHEⅡ)评分、院内死亡率及心肌肌钙蛋白(cTn)基线、最低、最高水平高于非T2MI组(P<0.05)。本组患者疾病类型以肺部疾病为主,占48.8%(140/287),T2MI组患者中重症肺炎、感染性休克、出血性疾病者占比高于非T2MI组,社区获得性肺炎(非重症)、多发伤者占比低于非T2MI组(P<0.05)。T2MI患者重度贫血、呼吸衰竭、多器官功能障碍综合征(MODS)、急性肾损伤(AKI)、休克及出血性事件发生率高于非T2MI组(P<0.05)。多因素Logistic回归分析结果显示,APACHEⅡ评分〔OR=1.070,95%CI(1.021,1.121)〕、呼吸衰竭〔OR=4.847,95%CI(2.533,9.277)〕、休克〔OR=5.922,95%CI(2.558,13.710)〕、出血性事件〔OR=7.963,95%CI(1.392,45.561)〕是ICU机械通气患者发生T2MI的独立影响因素(P<0.05)。结论本组ICU机械通气患者T2MI发生率为45.3%,APACHEⅡ评分、呼吸衰竭、休克、出血性事件是ICU机械通气患者发生T2MI的独立影响因素。     

Synthesis of Riboflavin Nucleotides by Mature Human Erythrocytes

Intact human red blood cells can synthesize FAD and FMN from riboflavin.The rate of synthesis of FAD is linearlyproportional to the concentration of riboflavin in the medium at levels below 0.9µM. With 0.9 µM riboflavin, the rate ofsynthesis is about 0.1 mµmole FAD/ml.red blood cells/hour. Incubation of redblood cells with riboflavin can result inincreased red cell glutathione reductaseactivity when the enzyme is measured inthe absence of added FAD. This indicates that the FAD concentration in thered cells increased during the incubation. The rate of incorporation of radioactive riboflavin into red blood cells isthe same whether the cells are suspendedin plasma or in a phosphate-saline-glucose medium. The time it takes for halfthe FAD in normal human red bloodcells to turn over is calculated to beabout 6 days, assuming a single mixingpool of red cell FAD.

Submitted on March 9, 1970 Revised on April 28, 1970 Accepted on May 8, 1970     

The Elevation of Adenosine-Triphosphate Levels in Human Erythrocytes

It has previously been possible to doublethe level of ATP in human erythrocytes byincubation of the cells at 37° for 10 hr withglucose and adenine. The present study describes a further increase in the ATP leveland some of the possible mechanisms involved. Addition of 5 mM pyruvate to amedium containing 32 mM inorganic phosphate, glucose, and adenine elevated thelevel of ATP threefold during a 10-hr incubation. Pyruvate could be replaced byinosine but the presence of both limitedthe elevation of ATP to twice that of freshcells. This limitation may be overcome bythe use of 96 mM phosphate in the incubation medium, in which case the intracellular level of ATP is tripled within 2 hr. Theconditions which limit the accumulation ofATP are associated with low intracellularphosphate concentrations and the accumulation of organic phosphates, especially, inthe presence of inosine, 2,3-diphosphoglycerate. Utilizing ¹⁴C-glucose labeled incarbons 1, 2, or 6, it has been shown thatwhen ATP is being rapidly elevated, thepentose moiety of the adenine nucleotidesis mainly supplied (about 80%) by oxidationof carbon 1 of glucose, catalyzed by thedehydrogenases of the hexosemonophosphate shunt. In the presence of pyruvatethis activity is doubled. Pyruvate reoxidizesNADPH formed by this pathway, sincelactic dehydrogenase has some specificitytowards the NADPH. The involvement ofthe dehydrogenases of the hexosemonophosphate shunt is illustrated by the useof erythrocytes deficient in glucose-6-phosphate dehydrogenase. Incubation ofthese cells for 5 hr with glucose andadenine results in only a slight increase inATP formation, and pyruvate has no additional effect. Addition of inosine, however,leads to the same increment in ATP levelsseen in normal cells. The ATP and 2,3-diphosphoglycerate levels in 6-wk preserved blood can also be increased to threetimes that of fresh cells by incubation withglucose, adenine, pyruvate, and inosine ina medium high in inorganic phosphate.

Submitted on January 26, 1973 Revised on March 27, 1973 Accepted on March 28, 1973