Hisashi Sanui

Measurement of inorganic orthophosphate in biological materials: Extraction properties of butyl acetate

Abstract The properties of several organic solvents were investigated to determine their suitability for use in phosphomolybdic acid (PMA) extraction procedures for the measurement of inorganic orthophosphate (P i ). Butyl acetate exhibited the most satisfactory properties for measurements at 310 nm, which is the absorption peak for unreduced PMA. Butyl acetate exhibits essentially no absorption at 310 nm, is highly selective for PMA over molybdate and silicomolybdate, does not extract molybdate in the presence of trichloroacetic acid (TCA), exhibits negligible volume changes when equilibrated with equal volumes of aqueous phase, and is among the least toxic of organic solvents. Optimal conditions of acidity, molybdate concentration, and reaction and extraction times were determined for the formation of PMA and extraction into butyl acetate. A procedure for measurement of P i in biological material employing 8% TCA for precipitation of organic material at 0°C, reaction of the supernatant with acid molybdate, extraction of the PMA with butyl acetate and reading of unreduced PMA at 310 nm is described. The procedure is simple, rapid, accurate, selective, and has high sensitivity. Because of the short (20 sec) sample exposure to acid molybdate, it is suggested that the procedure may also be useful in the measurement of P i in the presence of adenosine triphosphate such as for the assay of adenosinetriphosphatase activity.

Application of Atomic Absorption in the Study of Na, K, Mg, and Ca Binding by Cellular Membranes

Atomic-absorption spectrophotometry is applied in the analysis of biological membrane materials for micromolar levels of the physiologically important cations, Na, K, Mg, and Ca. Instrumental parameters and chemical interferences are investigated and a method for the measurement of all four cations in a single sample of ashed membrane material is formulated, tested, and applied. Results are highly satisfactory, and demonstrate that atomic-absorption spectrophotometry affords biologists a powerful and sensitive tool for the study of the important rôle played by inorganic ions in living systems.

Chemical and ionization interferences in the atomic absorption spectrophotometric measurement of sodium, potassium, rubidium, and cesium

Abstract Although chemical and ionization interferences significantly affect the atomic absorption signal of the alkali metals, suitable corrective measures permit accurate analysis of these elements. The observed interferences are affccted in opposite ways by flame temperature, chemical depression of absorption produced by anions decreasing, and ionization enhancement produced by cations increasing with increasing flame temperature. Anionic depression is small in an acetylene-air flame and moderately large in a propane-air flame, increasing in the sequence sulfate M phosphate depressing cesium absorbance approximately 40%. Conversely, ionization enhancement by cations is small in a propane-air flame and large in an acetyleneair flame, the effect on rubidium absorbance increasing in the sequence Mg M cesium producing a twoflod increase in absorbance. This is in the order of decreasing ionization potentials, indicating a direct relationship between ionization potential, degree of ionization, and enhancement produced. From consideration of the over-all effect of flame temperature on various interferences, we conclude that the propane-air flame is probably the most satisfactory for alkali metal analysis, especially for rubidium and cesium. Recovery studies on dialyzed and ashed rat liver microsomes and known controls demonstrate that addition of 15 m M lanthanum, to minimize anionic interferences, and addition of moderate concentrations of cesium, rubidium, or potassium, to minimize cationic enhancement, permit accurate and reliable measurement of the alkali metal cations in biological materials in the presence of potentially interfering cations and anions.

Changes of intracellular and externally bound cations accompanying serum stimulation of mouse balb/c 3t3 cells.

Abstract Serum stimulation of DNA synthesis in quiescent mouse BALB/c 3T3 cells is accompanied by changes in total and intracellular Na+, K+, Mg2+ and Ca2+ content and in externally bound divalent cations. A sharp increase in the rate of [3H]thymidine incorporation into DNA occurred after 10 h incubation following stimulation with 20% calf serum. This increase in DNA synthesis was accompanied by a decrease in intracellular Ca2+, little change in K+ and an increase in Mg2+ which reached 15% after 17 h. Incubation of confluent cultures for 17 h in media containing various serum concentrations caused the rate of DNA synthesis to increase rectilinearly with concentration, being 30-fold greater at 20 than at 0% serum. Concomitantly, intracellular cation changes occurred, levels at 20% serum as compared with 0%, being Na2+, 91%; K+, 103%; Mg+, 114%; and Ca2+, 41%. Externally bound divalent cations exhibited substantial serum dependence, bound Mg2+ at 20% serum decreasing to 45%, and Ca2+ to 22% of values at 0% serum. Results suggest that serum exerts its effects on cell growth at least in part by perturbing the plasma membrane, causing displacement of membrane divalent cations with a resultant change in membrane permeability and subsequently in cellular ion content. Although various cations including Na+, K+ and Ca2+ may be of significance in the regulation of cellular growth, our data from this and previous studies support the concept that intracellular Mg2+ is of primary importance in the control of cellular metabolism and growth.

Activated oxygen ashing of biological specimens for the microdetermination of Na, K, Mg, and Ca by atomic absorption spectrophotometry.

Abstract A procedure employing electrically activated oxygen for the oxidation of organic substances at relatively low temperatures (100–125°C) and requiring no large additions of chemical reagents is applied to the destruction of biological materials preliminary to elemental analyses by atomic absorption spectrophotometry. Results of recovery studies for micromolar levels of Na, K, Mg, and Ca in rat liver cell microsomes are highly satisfactory when specially designed quartz ashing chambers are used with the Tracerlab LTA-500A low temperature asher. Activated oxygen ashing appears to offer a unique and superior method for the treatment of milligram amounts of biological materials preliminary to elemental analyses of samples containing micromolar levels of biologically important elements.

Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells

Lead at slightly subtoxic concentrations markedly stimulated the rate of DNA synthesis in cultured animal cells. This stimulation was closely correlated with formation of a precipitate that was adsorbed and taken up by the cells under certain medium conditions. Data suggest that a precipitate-induced perturbation of the surface membrane leads to intracellular changes responsible for stimulation of DNA synthesis. Maximum stimulation of3H-thymidine incorporation by optimum concentrations of lead is delayed about 8 h compared to that in serum stimulation. In cells stimulated significantly by lead, but not in unstimu-lated cells, a reproducible rise of about 13% in intracellular magnesium occurred over a 24 h period, with an 8 h lag in the increase compared to that observed in serum stimulation. In view of the increases in intracellular magnesium consistently associated with and preceding stimulation of DNA synthesis by several different mitogens including serum and insulin, the present time-coordinated positive correlation between magnesium and DNA synthesis provides evidence for the primary involvement of this divalent cation in growth stimulation produced by lead.

An atomic absorption method for cation measurements in Kjeldahl digests of biological materials

Abstract Sulfate ion produced little or no interference in absorption by sodium, potassium, and magnesium, but produced a large depression in calcium absorbance in the atomic absorption spectrophotometric measurement of these cations in an acetylene-air flame. Nearly maximal depression of calcium absorbance by 2 m M sulfate was followed by a plateau region of only slight depression from 2 m M to 1 M sulfate concentration. Presence of 25 m M lanthanum in the samples resulted in no depression of calcium absorbance up to 2 m M sulfate, a sharp decrease to about 30 m M sulfate and a plateau from 30 m M up to 1 M sulfate. From these observations, it was determined that the addition of H 2 SO 4 to provide approximately 40 m M added sulfate in standards and samples permitted accurate measurement of calcium even though the original sample contained relatively high and variable sulfate. The results provided the basis for a practical method for the measurement of sodium, potassium, magnesium, and calcium in Kjeldahl digests of biological materials. The optimum condition for analysis was determined to be addition of reagents to give 25 m M lanthanum, 4 m M cesium, 300 m M hydrogen ion, and 40 m M sulfate in standards and samples for analysis in an acetylene-air flame by atomic absorption spectrophotometry. Application of this method to a study of the recovery of cations added to dialyzed rat liver homogenate demonstrated that sodium, potassium, magnesium, and calcium could be accurately measured in Kjeldahl digests, which could also be used for the measurement of total nitrogen.

Sodium and Potassium Binding by Microsomes from Nasal Salt Gland, Harder's Gland, Kidney and Liver of Sea Gull.

Summary Microsomes from the nasal salt gland, liver and kidney were capable of binding Na+ and K+ at a level of 1.1–1.6 meq/g N. Microsomes from Harders gland exhibited a Na+ and K+ binding of 2.3–2.5 meq/g N, which possibly represents binding to a mucopolysaccharide low in nitrogen. The high efficiency of the nasal salt gland could be associated with the elution-type mechanism of ion transport.