A Comparison of Normal Red Cell ATP Levels as Measured by the Firefly System and the Hexokinase System

Widely divergent normal red cell ATP levels have been reported by investigators using different methods. In order to clarify the cause of thesediscrepancies and to establish correct normal values for red cell ATP, the firefly technic for measuring ATP levels was compared with other methods. TheATP content of TCA filtrates of red cells was the same when determined bythe firefly method as by the hexokinase-linked technic. Relatively low concentrations of protein were found to stimulate light output when lyophilyzedfirefly extract, but not freshly prepared firefly extract, was used. Thus, falsely,high values were obtained when red cell extracts were examined, unless protein was also added to the standard. Storage of heparinized blood for as littleas 1 hour resulted in a substantial decrease in red cell ATP levels. The losswith ACD blood was less, and could be obviated entirely by using an ACDsolution with a pH adjusted to between 3.5 and 4.0. Removing the buffy coator washing cells in saline resulted in no further loss of red cell ATP. Extraction of washed red cells with TCA resulted in an average loss of 4.6 per cent ofATP, while extraction of whole blood with TCA resulted in a 14 per cent lossof ATP. In contrast, perchloric acid extraction resulted in no ATP loss. If ATPdeterminations are carried out using the firefly method, protein should beadded to the standard. If red cells must be stored for any period of time priorto extraction of ATP, an ACD solution with a pH of 3.5 to 4.0 should be used.If extracts of red cells are made, perchloric acid appears significantly superiorto trichloroacetic acid.

Submitted on January 10, 1967 Accepted on March 1, 1967     

Studies on Human Erythrocyte Nucleotide Metabolism. I. Monisotopic Methodologies

Methods were developed to assay theactivities of ribosephosphate pyrophosphokinase (RPK, PRPP synthetase, E.C. 2.7.6.1) and adenine phosphoribosyltransferase (APRT, AMPpyrophosphorylase, E.C. 2.4.2.7) inhemolysates of human erythrocytes.RPK activity was determined by measuring the production of phosphoribosylpyrophosphate (PRPP) and the formation of adenosine mononucleotide(AMP), the two products of the reaction. The K_m for adenosine triphosphate (ATP) was 0.12 mM with thePRPP method and 0.1 mM with theAMP method. The K_m for ribose-5-phosphate (R-5-P) was 0.14 mM withthe former method and 0.033-0.042 mMwith the latter. The pH optimum withthe PRPP method was between 7.6 and8.2. The mean RPK activity of hemolysates of normal mature erythrocyteswas 30.5 ± 3.78 µmoles of PRPP or30.6 ± 3.82 µmoles of AMP produced/hr per 10¹⁰ erythrocytes at 37°C. Although RPK activity of normalmature erythrocytes did not appear tobe markedly age dependent, activitywas substantially increased in reticulocyte-rich blood. APRT activity wasassayed by measuring the AMP produced in a system linked to the generation of oxidized nicotinamide adenine dinucleotide (NAD). The activityof normal mature erythrocytes was 3.38± 0.37 µmoles of AMP formed/hr per10¹⁰ erythrocytes at 37° C. Enzyme activity was significantly elevated inreticulocyte-rich blood and in the erythrocytes of a patient with the Lesch-Nyhan syndrome. These methods obviated the need for isotopic techniquesand were readily applicable to smallsamples of blood.

Submitted on August 30, 1971 Revised on November 15, 1971 Accepted on November 16, 1971     

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     

Preservation of Red Blood Cells: Studies of Erythrocyte Adenine Nucleotides Using Luminescence Analysis

The levels of ATP and total adenine nucleotides (ATP + ADP + AMP) were determined by firefly luciferase assay in red blood cells during storage for 5 weeks at 4 degrees C. With few exceptions, no significant differences in nucleotide levels were found between whole blood stored in CPD-adenine and various preparations of red blood cells in CPD-adenine or CPD with saline-adenine-glucose (SAG) as additive. The levels of ATP and total adenine nucleotides during storage are discussed in relation to glucose levels, extracellular pH and shelf life of the red blood cells.     

The In Vitro Restoration of Red Cell 2,3-Diphosphoglycerate Levels in Banked Blood

The demonstration that red cell levels of2,3-DPG play a central role in determining the affinity of hemoglobin for oxygenhas resulted in a renewed interest inmethods for maintaining or restoring thelevel of this organic phosphate in storedblood. The effects of addition of inorganic phosphate, inosine and pyruvate,individually or in various combinations,all in a final concentration of 10 mMwere evaluated 1 and 4 hours after supplementation of ACD-stored blood, 21 to28 days old. In 14 studies the initial2,3-DPG level averaged 176 mµmoles/ml. RBC. In normal fresh blood the2,3-DPG was 4200 ± 400 mµmoles/ml.RBC. Inosine addition raised the 2,3-DPG to 1395, inosine and phosphate to1528, inosine and pyruvate to 3363,while the combination of inosine, pyruvate and phosphate increased the level to6637 mµmoles/ml. RBC. After 2-3 hoursof incubation most of the 2,3-DPG restoration had occured. The maximum effects did not require prior pH correctionof the blood. Although inosine in a finalconcentration of 10 mM was requiredfor optimum effects, the phosphate andpyruvate concentrations could be reduced to 4 mM. In the presence ofinosine and phosphate alone the red cellaccumulated large quantities of triosephosphates, fructose diphosphate andglucose-6-phosphate. These levels werereduced by the addition of pyruvate.Pyruvate addition appears necessary toprovide sufficient NAD for maximum2,3-DPG synthesis. Associated with2,3-DPG restoration of the stored bloodthere was a rise in the P₅₀ (the oxygentension at which hemoglobin is 50%saturated) from a mean of 16.7 to 31.6mm. Hg.

Submitted on May 11, 1970 Revised on June 27, 1970 Accepted on July 23, 1970     

Release of Histamine from Human Basophils

A method is described for obtaining basophil-enriched preparations ofwhite cells from normal donor blood and from the blood of patients withchronic myeloid leukemia. This histamine content of basophils obtained fromnormal donor blood was found to be equivalent to 2.4 ± 0.6 µg. histamineacid phosphate/10⁶ basophils, and the histamine content of basophils obtainedfrom patients with chronic myeloid leukemia was found to be equivalent to1.8 ± 1.4 µg. histamine acid phosphate/10⁶ basophils. The difference betweenthe two mean values was significant (0.01 > p > 0.001).

Histamine was found to be present in the granule fraction of basophilsand was released from basophils in conditions associated with granule lysis.Human basophils were shown to be capable of active movement and were ableto phagocytose sensitized group A red cells. The basophils were less activeas phagocytes than neutrophils and eosinophils. Granule lysis occurred independantly of phagocytosis in the A anti-A immune system, in human serumabsorbed with zymosan, and following the addition to basophils of eosinophilperoxidase.

Submitted on January 24, 1966 Accepted on January 17, 1967     

A method for the measurement of red cell dimensions and calculation of mean corpuscular volume and surface area

1. A photomicrographic method was devised for the measurement of thedimensions of red cells in rouleaux in plasma. With normal blood valid conclusions were drawn from 80 cell measurements with a high degree of confidence.

2. The mean corpuscular volume and the surface area were computed fromthese dimensions by formulae which treated the cell as a spheroid.

3. The dimensions, mean corpuscular volume and surface area of normalhuman red cells were as follows: 8.28 µ diameter, 1.71 µ thickness, 82 µ³volume and 134 µ² surface area.

4. The computed mean corpuscular volume was in agreement with therefractometric determination.

Submitted on January 7, 1958 Accepted on April 12, 1958     

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     

The Carbohydrate Metabolism and Respiration of Isolated Small Lymphocytes: In Vitro Studies of Normal and Phytohemagglutinin Stimulated Cells

1. A homogeneous population of small lymphocytes with an average size of6.7 micra was isolated from equine blood.

2. These cells could be maintained in vitro, with essentially complete survivalfor 24 hours, and with a 50% viability for five days.

3. The small lymphocytes consumed glucose at a rate of 1.87 mµmoles/1 x10⁷ cells/minute, and produced lactic acid at a rate of 2.30 mµmoles/1 x 10⁷cells/minute.

4. The oxygen consumption of small equine lymphocytes was 1.023 ± .165mµmo1es oxygen 1 x 10⁷ cells/minute, and that of mixed peripheral bloodleukocytes, 1.25 ± .07 mµmoles oxygen/1 x 10⁷ cells/minute, as determined, using a Clark oxygen electrode.

5. Lymphocyte glycolysis was stimulated under anaerobic conditions (Pasteur effect), and viability appeared unimpaired after 24 hours in a N₂ environment.

6. Uncoupling of oxidative phosphorylation by the addition of 2,4, dinitrophenol stimulated both respiration and glycolysis.

7. The glycogen content of the normal small horse lymphocyte was 7.45 ±1.04 µg glucose equivalents per 1 x 10⁷ cells.

8. Many of the initial cell population were transformed into "blasts" followingthe addition of phytohemagglutin to the tissue culture medium. Thisresponse was associated with an increase in the rate of glycolysis andrespiration by 24 hours, and a rise in intracellular glycogen by 48 hours.

Submitted on December 7, 1966 Accepted on May 22, 1967     

A Direct Assay for Dihydrofolate Reductase in Human Leukocytes

A new direct spectrophotometric assay for dihydrofolate reductase hasbeen developed. The reaction is observed at 282 mµ and is based on thedisappearance of dihydrofolate. Based on this assay, the values for normalleukocytes are 0-5 mµmoles of dihydrofolate reduced to tetrahydrofolate,per mg. of protein per hour at 28 C. The respective values for chronicmyelogenous leukemic granulocytes are 25-67, chronic lymphocytic leukemia 0-18, acute lymphocytic leukemia 7-19, and for leukocytes of leukemoid reaction 4 to 5.

Submitted on October 2, 1963 Accepted on November 23, 1963     

Size Dependence of Ghosts From Stored Erythrocytes on Calcium and Adenosine Triphosphate

Heterogeneity within ghosts preparedfrom stored erythrocytes was studiedby multichannel particle analysis ofsize distributions of reconstitutedghosts. On restoration of isotonicity ofhemolysates from fresh erythrocytes,ghosts were smaller than the corresponding red cells. After subsequentincubation for 15 min, they swelled toa final stable volume. The hemolysisof stored erythrocytes and restorationof isotonicity yielded ghosts similar tothose from fresh cells, but, after subsequent incubation, a second population of small ghosts appeared. Thispopulation resulted from markedlydiminished ghost swelling, that is attributed to decreased sodium permeability and increased membranerigidity, causing resistance to changesin volume. Erythrocyte ATP depletionduring storage of ACD blood was associated with an increase in the percentage of small ghosts. Similar ghostswere produced from fresh cells whenCaCl₂ (1 x 10^-6 M) was introduced intoghosts during hemolysis. The erythrocyte calcium content increased duringprolonged storage from 11.3 ± 6.3 µgatoms/liter of cells (stored for 1-2 wk)to 22.0 ± 7.0 µg atoms/liter of cells,after 7-9 wk of storage. The introduction of nucleotides (2 x 10^-3 M) intoghosts during hemolysis of storedcells prevented the appearance ofsmall ghosts. The inhibitory effect wasgreater than predicted from stabilityconstants of calcium nucleotide complexes, indicating the specific interaction of nucleotides with the membrane. The percentage of small ghostsfrom red cells stored for various timesagreed with the percentage of nonviable cells derived from the literature. It is concluded that the decreasein ATP and the accumulation of calcium in stored erythrocytes induceconformational changes of membranefibrous (contractile) proteins that results in decreases in membrane elasticity and permeability, which is inturn reflected by formation of smallghosts.

Submitted on September 20, 1971 Revised on February 23, 1972 Accepted on February 25, 1972     

Ferritin-Containing Bodies in Human Small Intestinal Epitheliuin

Ferritin was identified in the absorptive cells of human jejunum from normaliron-replete subjects by the demonstration of the tetrad form of the iron hydroxide micelle of the ferritin molecule. In a few sections ferritin moleculeswere observed to be dispersed in the cytoplasm, but usually they were withininclusion bodies found in the apical cytoplasm. These ferritin-containing bodieswere oval in profile and 0.5-1.5 µ long. They were not bounded by membranebut did have a moderately dense background substance which made a sharpboundary with the cytoplasmic ground substance. A few clues suggest thatthe body itself, but not necessarily its substance, is formed within the Golgiapparatus.

Submitted on February 12, 1963 Accepted on May 27, 1963     

Energy Metabolism in Human Platelets:Interrelationship Between Glycolysis and Oxidative Metabolism

Glycolysis and oxidative metabolismwere assessed in washed human platelets incubated in a Ca⁺⁺ and Mg⁺⁺ freeKrebs-Ringer bicarbonate buffer, pH7.4 at 37° C for 1 hour. Glycolytic ratewas 45-65 per cent lower under aerobicthan anaerobic conditions. Glycolysiswas decreased further when albuminwas present in the incubation mediumand under these conditions glucose uptake, glycogen utilization and lactateproduction were 21.4, 15.8 and 78µmoles/hr. per 10¹¹ platelets, respectively. The oxidation of 6-¹⁴C glucosewas 0.39 µmoles/hr. per 10¹¹ plateletsand of U-¹⁴C palmitate 0.19 µmoles/hr.per 10¹¹ platelets, and the rate of oxidation of either substrate was increased inthe absence of the other. The uncoupling agent, dinitrophenol, stimulatedoxidation of glucose to a much largerextent than fatty acid. It is concludedthat both glycolysis and oxidative phosphorylation are important to plateletenergy metabolism, that either pathwaymay compensate for decreased activityof the other and that, under conditionsof metabolic stress, glucose is preferredto fatty acids as a substrate for oxidativemetabolism.

Submitted on December 19, 1969 Revised on February 11, 1970 Accepted on February 20, 1970     

Red cell thickness in normals and in pernicious anemia

A method previously described by Williams and Wyckoff, Bessis, and othersis adapted for measuring the thickness of the dried blood cell. Statistical analysisof the results reveals the presence in pernicious anemia of a distinct type of cellwhich in the dried blood film has a mean diameter 1 µ above normal and athickness 0.224 µ less than normal. These pathologic cells, which thus flattenout when spread on a glass slide, also shrink more than do normal cells whendried in a blood film.

Submitted on May 16, 1952 Accepted on June 24, 1952     

Porphyria Cutanea Tarda Associated with Chronic Granulocytic Leukemia Treated with Busulfan (Myleran)

A patient with chronic granulocytic leukemia terminating in "blast crisis"and marked porphyria is presented. Quantitative porphyrin studies revealed:urinary uroporphyrin 8894 to 11,453 µg./24 hours, urinary coproporphyrin 107-486 µg./24 hours, fecal coproporphyrin 55-116 µg./Gm. dry weight and fecalprotoporphyrin 22-38 µg./Gm. dry weight. A review of the clinical aspects ofporphyria is briefly presented, together with some speculation as to the etiologyof the porphyria in the present case. The possibility is present that the busulfantherapy either initiated the disorder or triggered its development from a previously occult state. If so, this would be another manifestation of busulfantoxicity, of which pigmentation is the most common.

Submitted on September 13, 1963 Accepted on November 17, 1963     

On the Distribution of Red Cell Volumes

A large number of red cell volume distribution curves can be generated onthe same sample of blood by manipulating the length of the Coulter Counteraperture and the shape of the red cell. Curves derived from sphered red cellsflowing through an aperture 70 x 98µ long, or longer appear to be relativelyfree from artefacts and therefore the most likely to be "real."

The use of long apertures to size sphered red cells will give a volume distribution curve that is approximately Gaussian and with a mean that is largerelative to the variance. Whether red cells are Gaussian or log normal in theirvolume distribution is very difficult to determine. On a particle populationwhere volumes are controlled as closely as on red cells (i.e. with such a lowvariance), the normal and the log normal distributions become practicallyindistinguishable. If in the future this decision can be reached by some independent means, then the volume distributions using the Coulter volume transducer can be easily and simply fitted to the shape desired by appropriatemanipulation of aperture length. These ideal volume distribution curves arenot likely to differ markedly from those obtained under the conditions suggested in this paper. For the present, use of a 70 x 98µ aperture to size redcells sphered by suspension in isotonic Sodium Potassium Tartrate will contribute to considerably greater accuracy in identification of red cell subpopulations in abnormal blood.

Submitted on July 28, 1967 Accepted on October 7, 1967     

Report of a Three-Year Study of Red Cell Mobility and the Slowing Factor in Serum of Persons with Lymphoma

1) A variety of pathologic conditions retard the electrophoretic mobilityof the red blood cell.

2) Two retarding factors have been found in the blood serum, one that reduces the mobility of the red cell from normal (1.33µ/cm./sec./volt) to 0.89µand one that reduces it to 1.14µ.

3) The "0.89" factor and the "1.14" factor can be differentiated and identified.

4) The 0.89-factor has been found in the serum of 99 per cent of patientswith cancer and Hodgkin’s disease studied and has been lacking from theserum of all persons with benign tumor.

5) The 0.89-factor has also been found in patients with infectious mononucleosis.

6) A few physical characteristics that enable differentiation of the two slowing factors are described.

7) Clarification of the role played by the 0.89-factor in malignant neoplasmis deemed important.

8) Determination of the presence or absence of the 0.89-factor in the bloodserum may become of value in differential diagnosis of malignant neoplasm.

Submitted on May 4, 1962 Accepted on July 18, 1962     

Studies on Hemoglobin: 1. Antigenic Properties of Human, Canine and Rabbit Hemoglobin Solutions

Specific antisera with high antibody titers could be prepared against adult,cord blood, dog and rabbit hemoglobin solutions. These sera were employedto study the antigenic properties of the above hemoglobins.

Common antigenic properties could be detected between hemoglobin solutions prepared from human, canine and rabbit blood.

No immunologic difference could be found between the fetal fractions ofadult human blood, cord blood and thalassemia patients’ hemoglobin solutions.

Submitted on February 26, 1963 Accepted on July 19, 1963     

Radioactive vitamin B12 absorption studies: results of direct measurement of radioactivity in the blood

A new method for the determination of the absorption of vitamin B₁₂ has beendescribed using measurement of radioactivity in the blood or plasma after theingestion of physiologic test doses of Co⁶⁰ labeled vitamin B₁₂.

Although doses of 1.0 µc. (0.92 µg. vitamin B₁₂) gave higher counts, equallyreliable results were obtained with 0.5 µc. (0.46 µg. vitamin B₁₂). The radioactivitywas found in the plasma portion of the blood.

With this method it was possible to differentiate between all of nine patientswith pernicious anemia and 36 control subjects.

In non-pernicious anemia subjects and in pernicious anemia patients givenintrinsic factor, there was a relatively delayed rise in the blood or plasma radioactivity until a peak was reached in the 8 to 12 hour interval after the ingestionof the test dose. This absorption curve was quite different from the early rise observed by others after massive oral doses of vitamin B₁₂, indicating a differentmode of absorption.

Following the peak blood concentration the radioactivity gradually declinedand small amounts usually persisted for as long as one week, quite different fromthe rapid disappearance after parenteral administration previously reported.

This method appears valuable in the diagnosis of pernicious anemia and othervitamin B₁₂ malabsorptive states, in the evaluation of intrinsic factor activity,and in studies of various aspects of the metabolism of vitamin B₁₂.

Submitted on June 25, 1956 Accepted on August 12, 1956     

Red Cell Preservation: Further Studies with Adenine

Supplementation of the ACD-preservative with small amounts of adenine(0.5 µM per ml. amounting to 37 mg. of the base or 56 mg. of adenine sulfateper 550 ml. unit of blood) preserved satisfactory viability (post-transfusionsurvival greater than 70 per cent) of stored human red cells for 5 to 6 weeks.In the concentrations used, the addition of guanine, cytidine or uridine, aloneor in combination with adenine, had little or no effect in extending viability.Hypoxanthine, even in large amounts, did not appear to be toxic to thestored cells. Preservation of viability after 6 weeks of refrigerated storagemay be somewhat improved by storage in certain plastic containers as compared with glass.

Submitted on April 16, 1962 Accepted on June 22, 1962