FACTORS INFLUENCING THE RESPIRATION OF ERYTHROCYTES : II. MAMMALIAN RETICULOCYTES

1. The respiration of the reticulocytes of the rabbit has been measured during the period of an anemia produced by phenylhydrazine. Though the respiration increased greatly during the phase of regeneration, the oxygen consumption per billion reticulocytes throughout the period remained approximately the same. 2. The respiration of the reticulocytes was affected by changes in the reaction of the medium in which they were suspended, and was at its maximum about a pH of 8, with a probable intracorpuscular pH of about 7.75. 3. Variations in the tonicity of the suspending medium did not produce any great change in the respiration of the reticulocytes. 4. The presence of glycine, alanine, and glucose in the suspending medium resulted in no acceleration in the respiration of the cells. At higher concentrations glucose tended to depress the respiration. The material oxidized appears to be mainly or entirely contained in the corpuscles at the time they are liberated from the marrow. 5. A comparison is made of the respiration of the reticulated nucleated red cells present in the blood of anemic fowls and the non-nucleated reticulated red cells of rabbits. On the basis of equal volumes of cells, the respiration of the former is about twice that of the latter, while this in turn is about six times as great as the nucleated but non-reticulated normal red cells of the fowl.     

THE EFFECT OF LOW OXYGEN TENSION ON TISSUE METABOLISM (RETINA)

Lactic acid production by rat retina in a medium containing phosphate was studied chemically. One half as much lactic acid was found as in a medium containing bicarbonate. In our experience the rate of respiration in a phosphate medium was sensitive to oxygen tension, for it was 38 per cent lower at 10 per cent and 51 per cent lower at 5 per cent oxygen than at 100 per cent oxygen. Previously Laser had reported no decrease in respiration at 5 per cent oxygen in phosphate medium. In phosphate medium, when the oxygen tension was varied, respiration and glycolysis bore a reciprocal relationship to each other. In bicarbonate medium, when the oxygen tension was lowered from 95 per cent to 5 per cent there was no significant change in the respiration, but glycolysis was increased nearly to the anaerobic level. This agrees with the earlier experiment of Laser in bicarbonate medium and adds support to his conclusion that the rate of glycolysis is controlled by oxygen tension rather than by the rate of respiration, under the conditions of the experiment.     

THE EFFECT OF CARBON DIOXIDE TENSION ON TISSUE METABOLISM (RETINA)

The metabolism of rat retina was found to be sensitive to the concentration of the carbon dioxide-bicarbonate buffer system. Increasing the carbon dioxide from 1 per cent to 5 per cent at constant pH nearly doubled both respiration and glycolysis. Increasing the carbon dioxide at constant pH from 5 per cent to 20 per cent had no effect on glycolysis, but depressed the Q _OO2 from 31 to 19. In a medium containing glucose and the 1 per cent carbon dioxide-bicarbonate buffer, the addition of succinate increased the Q _OO2 from 12 to 26, without affecting glycolysis. In a medium containing glucose and phosphate, succinate had no significant effect.     

A simple method for determination of red blood cell mechanical fragility in the rat

A method for measuring the mechanical fragility of red blood cells suitable for use in small laboratory animals, such as rats, is reported because of lack of such data in the literature. Whole blood is mixed with phosphate buffered saline in a tube containing glass beads. The tubes are rocked for 90 minutes, centrifuged and the percent hemolysis determined. Varying the osmolality of the saline suspending medium had little effect on the mechanical fragility of rat red cells prior to the NaCl concentrations at which a significant change in osmotic hemolysis occurred. The duration of rocking increased the mechanical fragility. Varying the pH (6.4-8.0) had no effect. The size of the glass beads changed the mechanical fragility as did varying temperature. The mean mechanical fragility of rat red blood cells was 46% hemolysis (80 adult male animals). Because of the small volume of blood required with this method, mechanical fragility of red cells of other small laboratory animals also may be determined.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

Metabolic effects of dehydroabietic acid on rainbow trout erythrocytes

Oxygen consumption and ATP concentration were measured in rainbow trout erythrocytes incubated in a physiological saline containing 0, 5, 15, 30 or 60 mg/l dehydroabietic acid. DHAA caused a decrease in cellular ATP level and oxygen consumption at concentrations above 15 mg/l. Haemolysis increased markedly, when the cellular ATP concentration decreased below 1 mM. These data suggest that increased breakdown of red cells may be the primary reason for jaundice which is observed in resin acid-toxicated fish.     

The influence of adenosine on intermediary metabolism of isolated hepatocytes

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37°C. This increased the concentration of the adenylic nucleotides ATP (+ 57 per cent), ADP (+ 39 per cent). Cyclic AMP was increased (+ 50 per cent) and the intracellular inorganic phosphate decreased (− 22 per cent). These changes were accompanied by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bisphosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD⁺/NADH ratio along with the increase of the ATP/ADP × PO₄ ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD⁺/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.     

Glucose influence on germ tube production inCandida albicans

The influence of different glucose concentrations was tested in a minimal synthetic medium onCandida albicans strain. After 18 hours of starvation, germ-tube (GT) production, amount of consumed glucose, oxygen and the pH of the medium were checked every hour from the beginning through the end of the experiment. Optimal GT production was obtained with 1 g/l of glucose. At this concentration the greatest glucose and oxygen consumption were also noted. No pH variations in the medium were observed in all of the glucose concentrations used. At 3 and 5 g/l glucose concentrations a lower GT production were obtained. The Crab-tree effect might interfere with GT production when glucose concentration is higher than 1 g/l. This data may support the hypothesis that GT production is strictly glucose dependent.     

Iodination-Coupled Tetrazonium and Ferric Ferricya-Nide Reduction Stains for Differentiating Red Blood Cells Containing Fetal or Adult Hemoglobin

Either the iodination-coupled tetrazonium reaction or the ferric ferricyanide reduction procedure can be used to differentiate red blood cells containing fetal hemoglobin (hemoglobin F) from those containing adult hemoglobin (hemoglobin A) in blood smears. Oxalated blood is diluted with 3 parts of physiological saline, and smears are made on slides. The air-dried slides are treated with absolute ethanol for 2 min, dried, and placed in phosphate-citrate buffer of pH 3.2-3.6 for 1 min at 37°C. They are then rinsed in distilled water, and dried for storage or stained at once by either the iodination-coupled tetrazonium or the ferric ferricyanide reduction procedure. Adult hemoglobin is extracted by the buffer, so that red blood cells containing fetal hemoglobin give a much darker stain than those containing adult hemoglobin. The hemoglobin S of patients with sickle-cell anemia behaves like adult hemoglobin.     

Osmoregulation in the Halotolerant Alga Asteromonas gracilis

Asteromonas gracilis, a green wall-less halotolerant alga, grows on salt concentrations from 0.5 molar NaCl (seawater) to saturation (4.5 molar NaCl). The specific growth rate was maximal at concentrations between 0.5 and 2.5 molar and only gradually decreased above 2.5 molar. Photosynthetic oxygen evolution was maximal over a range of salinities around 2.5 molar and the photosynthesis to respiration ratio showed a maximum at 1.5 molar NaCl. The alga accumulates large amounts of intracellular glycerol in response to saline conditions. The glycerol content of the cells varied in direct proportion to the extracellular salt concentration, being about 50 and 400 picograms glycerol per cell in algae grown at 0.5 and 4.5 molar NaCl, respectively. In salt concentrations lower than 3.5 molar and at growth temperatures below 40 C, essentially all the glycerol was intracellular. Above 3.5 molar NaCl, about 25 per cent of the total glycerol leaked slowly from the cells to the medium. Treating the algae for several minutes at temperatures exceeding 47 C caused 50 per cent release of the internal glycerol. At 60 C, 100 per cent of the glycerol was released. When the extracellular salt concentration was increased or decreased, the intracellular glycerol varied accordingly, reaching its new intracellular level after a few hours. Both photosynthesis and respiration were inhibited on transfer of the cells from 1.5 to 3.5 molar NaCl but were not inhibited on transfer of the cells from 3.5 to 1.5 molar NaCl. The kinetics of photosynthetic resumption preceded the kinetics of glycerol biosynthesis. The above results indicate the existence of osmotic regulations in Asteromonas gracilis via the accumulation of intracellular glycerol.     

Rate of oxygen consumption by isolated articular chondrocytes is sensitive to medium glucose concentration

Cartilage tissue engineering typically involves the culture of isolated chondrocytes within a scaffold material. The oxygen tension within the engineered tissue is known to be an essential parameter for implant success. This will be sensitive to the oxygen consumption behavior of the embedded chondrocytes, which remains to be characterized. We report that the oxygen consumption of bovine articular chondrocytes is sensitive to glucose deprivation below 2.7 mM, increasing from a basal level of 9.6x10(-16) to <18.4x10(-16) mol/cell.h in 1.3 mM glucose. Further studies examined the influence of selecting high (18.4 mM) or low (5.1 mM) glucose medium on the oxygen tension in 2 mm thick cellular agarose constructs. A relative upregulation of oxygen consumption was observed in constructs cultured in low glucose medium. This resulted in the near-anoxic oxygen concentration of 5 microM oxygen in constructs seeded with 40x10(6) cells/ml, compared to 57 microM in the corresponding high glucose culture. The upregulation of oxygen consumption generally corresponded to the inhibition of glycolysis, which is consistent with the Crabtree phenomenon. Medium osmolarity (316-600 mOsm) had minimal effects on chondrocyte oxygen consumption rate. In conclusion, glucose availability is a critical parameter that regulates the oxygen tension within tissue engineered constructs.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VI. OBSERVATIONS ON THE GLYCOGEN CONTENT,CARBOHYDRATE CONSUMPTION,LACTIC ACID PRODUCTION,AND AMMONIA PRODUCTION OF EGGS OF ARBACIA PUNCTULATA

1. Under the present conditions of experiment, Arbacia eggs were found to contain an average of 110 mg. of acid-hydrolyzable carbohydrate (calculated as glucose) per gm. of egg protein. This carbohydrate was almost all in the egg proper, little or none being found in the jelly. To permit conversion of the data to other bases of reference the relation of nitrogen content to wet and dry weight and to egg number were determined. The eggs were found to contain 23.9 per cent solids, 0.10 mg. nitrogen per mg. dry weight, and 5.93 mg. nitrogen per 10⁶ cells. From these results, about 7 per cent of the egg dry weight is acid-hydrolyzable carbohydrate and about 65 per cent is protein. 2. Approximately one-half of the total acid-hydrolyzable carbohydrate was isolated in the form of an alkali-stable, alcohol-precipitable carbohydrate. This substance gave a typical glycogen color test with iodine, yielded glucose on acid hydrolysis, and had, within the limits of experimental error, the same optical rotation as glycogen from other animal sources. Since known amounts of glycogen were completely recovered when carried through the isolation process, the nature of one-half of the acid-hydrolyzable carbohydrate of Arbacia eggs remains undetermined. 3. In order to gain some estimate of the extent to which Arbacia eggs utilize their total carbohydrate for development, determinations of the oxygen consumption, respiratory quotient, carbohydrate consumption, lactic acid production, and ammonia production were made. While all samples of eggs were found to utilize carbohydrate from the 15th to the 24th hours of development at 20°C., certain samples of eggs consumed little or no carbohydrate from the 1st to the 6th hours, the period during which cell division proceeds most rapidly. In a number of instances where carbohydrate breakdown was lacking, a substantial proportion of the oxygen consumption could be accounted for on the basis of processes involving oxidation of protein or protein breakdown products.     

The influence of adenosine on intermediary metabolism of isolated hepatocytes.

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37 degrees C. This increased the concentration of the adenylic nucleotides ATP (+57 per cent, ADP (+39 per cent). Cyclic AMP was increased (+50 per cent) and the intracellular inorganic phosphate decreased (-22 per cent). These changes were accompaned by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bis-phosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD+/NADH ratio along with the increase of the ATP/ADP X PO4 ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD+/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.     

Glucose Respiration in Zygorhyncus moelleri; the Entry of Glucose into the Cells

When glucose was added to carbohydrate-starved cells of Zygorhyncus,moelleri the rate of oxygen uptake did not immediately riseto a constant value, but there was a lag period of 2 or 3 hoursbefore it reached its maximum level. The length of this lagperiod increased from a few minutes for short periods of starvationto 2–3 hours after 12 hours in a carbohydrate-free medium.Factors believed to affect cellular permeability (a cationicdetergent, adjustments of the pH, and of the potassium/calciumratio) reduced the length of the lag period by not more than40 per cent. of the original value without affecting the finalrate of oxygen uptake. Investigation of the entry of glucoseinto the cells showed that the rate of oxygen uptake was notlimited by the concentration of intra-cellular glucose for morethat about 11 per cent. of the lag period in starved cells.The reasons for this difference in the percentage of the lagperiod apparently due to a permeability barrier are tentativelydiscussed in connexion with the route by which glucose entersthe cells.     

THE METABOLISM OF TISSUE CULTURES : I. PRELIMINARY STUDIES ON CHICK EMBRYO

1. The metabolism of chick embryo tissues has been followed by analysis of the culture media after various periods of incubation in roller bottles. 2. The initial rate of glucose utilization is increased by increasing glucose in the medium from 100 to 500 mg. per cent. Total glucose used can be increased in the same way or by daily addition of small amounts. Glucose is used in greatest amount when the medium containing 100 mg. per cent is replaced daily. 3. Although glucose consumption appears necessary for survival of cultures it may be used at a rate far in excess of that required for life and maximal growth. Complete blocking of mitosis by colchicine does not alter the rate of glucose utilization. 4. Proteolytic activity of the cultures is shown by an increase in the amino nitrogen of the peptone medium after incubation with tissue. 5. Utilization of nitrogen from an amino acid medium is shown by a decrease in the amino nitrogen of this medium. Cells obtaining their nitrogen from amino acids proliferate as rapidly as those grown in a medium identical except for the substitution of peptone, but the cell type is markedly different, in that embryo muscle forms cells resembling regenerating adult muscle. 6. Lactic acid was formed in both the presence and absence of glucose. Its formation increased with increased glucose utilization. There is some evidence that lactate may be utilized, and that it favors growth in the absence of glucose. 7. Added pyruvate was rapidly metabolized by the tissues. It, too, favors growth slightly in the absence of glucose.     

Effect of glucose concentration on the rate of fructose consumption in native strains isolated from the fermentation of Agave duranguensis

Studies on hexose consumption by Saccharomyces cerevisiae show that glucose is consumed faster than fructose when both are present (9:1 fructose to glucose) in the medium during the fermentation of Agave. The objective of this work was to select strains of S. cerevisiae that consume fructose equal to or faster than glucose at high fructose concentrations by analyzing the influence of different glucose concentrations on the fructose consumption rate. The optimal growth conditions were determined by a kinetics assay using high performance liquid chromatography (HPLC) using 50?g of glucose and 50?g of fructose per liter of synthetic medium containing peptone and yeast extract. Using the same substrate concentrations, strain ITD-00185 was shown to have a higher reaction rate for fructose over glucose. At 75?g of fructose and 25?g of glucose per liter, strain ITD-00185 had a productivity of 1.02 gL^?1?h^?1 after 40?h and a fructose rate constant of 0.071?h^?1. It was observed that glucose concentration positively influences fructose consumption when present in a 3:1 ratio of fructose to glucose. Therefore, adapted strains at high fructose concentrations could be used as an alternative to traditional fermentation processes.     

Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture

Ammonia and lactate are the major byproducts from mammalian cells grown in medium containing glutamine and glucose. Both can be toxic to cells, and may limit the productivity of commercial bioreactors. The transient and steady-state responses of hybridoma growth and metabolism to lactate and ammonia pulse and step changes in continuous suspension culture have been examined. No inhibition was observed at 40 mM lactate. Cell growth was inhibited by 5 mM ammonia, but the cells were able to adapt to ammonia concentrations as high as 8.2 mM. Ammonia production decreased and alanine production increased in response to higher ammonia concentrations. Increased ammonia concentrations also inhibited glutamine and oxygen consumption. The specific oxygen consumption rate decreased by an order of magnitude after an ammonia pulse to 18 mM. Under these conditions, over 90% of the estimated ATP production was due to glycolysis and a large fraction of glutamine was converted to lactate.     

Regulation of 5-phosphoribosyl 1-pyrophosphate and of hypoxanthine uptake and release in human erythrocytes by oxypurine cycling.

Uptake and release of purines by red blood cells has been shown to be markedly sensitive to changes in pH, inorganic phosphate (Pi), and oxygen concentration (Berman, P., Black, D., Human, L., and Harley, E. (1988) J. Clin. Invest. 82, 980-986). The mechanism of this regulation has been further studied. We have shown that incubation of red cells in medium containing xanthine oxidase rapidly and completely depletes intracellular hypoxanthine and causes accumulation of 5-phosphoribosyl 1-pyrophosphate (PRPP) at physiological Pi concentrations. Hypoxanthine release from intracellular IMP is strictly dependent on PRPP depletion, induced by either alkalinizing the cells or by adding excess adenine. Xanthine oxidase abolishes this dependence. Oxygen depletion enhances adenine uptake and prevents hypoxanthine release. The results suggest that hypoxanthine release is governed by PRPP-dependent recycling of hypoxanthine to IMP. We propose that PRPP accumulation in red cells is regulated by a substrate cycle, comprising hypoxanthine, IMP, and inosine. Cycle flux is controlled by Pi inhibition and 2,3-bisphosphoglycerate activation of purine-5'-nucleotidase, which converts IMP to inosine. Oxypurine cycling may account for the sensitive control of purine uptake and release by changes in pH and oxygen tension that occur physiologically.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.