Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A™ column with mobile phase consisting of 0.05 M citric acid–0.1 M dipotassium hydrogen phosphate (pH 4.0)–methanol (97+3). The detection limits were 100–1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2–22.2 pmol/ml), ADP (5.5–22.2 pmol/ml), AMP (0.8–3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.     

Simple assay of 0.1–1.0 pmol of ATP,ADP, and AMP in single somatic cells using purified luciferin luciferase

The sensitivity of ATP determinations with crude firefly luciferin luciferase is limited by contaminating ATP converting enzymes, which cause a rapid decrease of the ATP level during the assay. Purified luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration. Sensitivity and specificity of the ATP assay are, therefore, considerably increased when purified enzyme is used instead of crude extracts of the enzyme. ATP, 0.1–1.0 pmol as well as higher amounts can be determined with commercial preparations of purified and stabilized luciferase. In ADP and AMP measurements with the luciferase assay, problems are arising from the enzymes required for the conversion to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides. Exclusion of contaminated enzymes and removal of ammonium sulfate from adenylate kinase were the only prerequisites for determinations of 0.1–1.0 pmol of ADP and AMP with purified luciferase. The application of the assay in determinations of ATP, ADP, and AMP in single preimplantation mouse embryos is described.     

Validation of a rapid, large-scale assay to quantify ATP concentration in spermatozoa

Quantification of ATP content in spermatozoa is a useful assay for evaluating sperm function; however, most detection methodology relies on assessing single samples. We have developed and validated a highly repeatable assay that permits simultaneous measurement of up to 78 samples. A key feature of this assay includes combination of a phosphatase inhibition and ATP extraction step that permits maximal detection of ATP and sample storage at -20 degrees C prior to assay. The assay was validated for spermatozoa from three different species, including turkey, rooster and boar. The sensitivity of the assay differed between avian and mammalian spermatozoa, with 2.5 x 10(6) spermatozoa being the lowest number of turkey and rooster spermatozoa that could be assayed compared to 2.5 x 10(5) boar spermatozoa. Concentrations of ATP in fresh turkey semen ranged from 2.14 to 15.6 nmol/10(9) spermatozoa; similarly, freshly collected rooster semen contained from 2.16 to 21.4 nmol ATP/10(9) spermatozoa. Evaluation of turkey semen that had been stored at 4 degrees C for 24 h revealed a decline in ATP concentrations (2.35 +/- 0.34 nmol ATP/10(9) spermatozoa). Likewise, cryopreserved rooster spermatozoa contained lower concentrations of ATP (0.05 +/- 0.01 nmol ATP/10(9) spermatozoa) than non-stored spermatozoa. Boar spermatozoa contained similar concentrations of ATP, whether fresh (74.2 +/- 8.1 pmol ATP/10(6) spermatozoa), stored for 1 day (77.0 +/- 8.1 pmol ATP/10(6) spermatozoa) or 5 days (81.96 +/- 8.1 pmol ATP/10(6) spermatozoa). For all three species, assay variation was low (inter-assay, 0.66-1.9% CV; intra-assay, 1.3% CV).     

Determination of picomole quantities of acetylcholine and choline in physiologic salt solutions

An assay capable of detecting tens-of-picomole quantities of choline and acetylcholine in milliliter volumes of a physiological salt solution has been developed. Silica column chromatography was used to bind and separate 10–3000 pmol [¹⁴C]choline and [¹⁴C]acetylcholine standards made up in 3 ml of a bicarbonate-buffered Krebs-Ringer solution. The silica columns bound 95–98% of both choline and acetylcholine. Of the bound choline 84–87% was eluted in 1.5 ml of 0.075 n HCl, whereas 95–98% of the bound acetylcholine was eluted in a subsequent wash with 1.5 ml of 0.030 n HCl in 10% 2-butanone. Vacuum centrifugation of the eluants yielded small white pellets with losses of choline and acetylcholine of only 1%. Dried pellets of unlabeled choline and acetylcholine standards were assayed radioenzymatically using [γ-³²P]ATP, choline kinase, and acetylcholinesterase. The net disintegrations per minute of choline[³²P]phosphate product was proportional to both the acetylcholine (10–3000 pmol) and choline (30–3000 pmol) standards. The “limit sensitivity” was 8.5 pmol for acetylcholine and 11.4 pmol for choline. Cross-contamination of the choline assay by acetylcholine averaged 1.3%, whereas contamination of the acetylcholine assay by choline averaged 3.1%.     

A bioluminescence method for direct measurement of phosphodiesterase activity

We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of <1 μU (pmol/min) of PDE activity in cell extracts containing 0.25–10 μg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible, and accurate method for quantifying PDE activities in small biological samples.     

A fluorescent aptasensor for analysis of adenosine triphosphate based on aptamer–magnetic nanoparticles and its single‐stranded complementary DNA labeled carbon dots

A new fluorimetric aptasensor was designed for the determination of adenosine triphosphate (ATP) based on magnetic nanoparticles (MNPs) and carbon dots (CDs). In this analytical strategy, an ATP aptamer was conjugated on MNPs and a complementary strand of the aptamer (CS) was labeled with CDs. The aptamer and its CS were hybridized to form a double helical structure. The hybridized aptamers could be used for the specific recognition of ATP in a biological complex matrix using a strong magnetic field to remove the interfering effect. In the absence of ATP, no CDs–CS could be released into the solution and this resulted in a weak fluorescence signal. In the presence of ATP, the target binds to its aptamer and causes the dissociation of the double helical structure and liberation of the CS, such that a strong fluorescence signal was generated. The increased fluorescence signal was proportional to ATP concentration. The limit of detection was estimated to be 1.0 pmol L^–1 with a dynamic range of 3.0 pmol L^–1 to 5.0 nmol L^–1. The specific aptasensor was applied to detect ATP in human serum samples with satisfactory results. Moreover, molecular dynamic simulation (MDS) studies were used to analyze interactions of the ATP molecule with the aptamer.     

Reactivating tammar wallaby blastocysts oxidize fatty acids and amino acids.

The tammar wallaby, Macropus eugenii, has a ruminant-like digestive system which may make a significant concentration of amino acids and fatty acids available to the blastocyst via uterine fluids. Fluorescent and radioisotope analyses were performed to determine the rate of glutamine and palmitate use by blastocysts recovered on day 0, 3, 4, 5 and 10 after reactivation induced by removal of pouch young (RPY). Between day 0 and 4 glutamine uptake increased from 15.6 +/- 6.6 to 36.1 +/- 2.7 pmol per embryo h-1 (P < 0.01) and ammonium production increased from 8.2 +/- 4.3 to 26.6 +/- 3.0 pmol per embryo h-1 (P < 0.01). Glutamine oxidation did not increase until day 10 after RPY (P < 0.01), but the percentage of glutamine oxidized increased from 4.5 +/- 3.1% during diapause to 31.2 +/- 12.6% (P < 0.01) by day 5 after RPY and increased further to 51.0 +/- 15.8% (P < 0.01) by day 10 after RPY. Palmitate oxidation also increased from 0.3 +/- 0.1 by day 0 blastocysts to 3.8 +/- 1.7 pmol per embryo h-1 (P < 0.01) by day 4 blastocysts. This increase provides a greater potential for ATP production, possibly to supply increased demand due to the coincident resumption of mitoses. The ATP:ADP ratio within blastocysts had reduced by the time of the first measurement at day 3 (0.5 +/- 0.2 pmol per embryo h-1; P < 0.01) compared with day 0 blastocysts (1.4 +/- 0.3 pmol per embryo h-1). It is likely that metabolism of amino acids and fatty acids contributes to the energy supply during reactivation of tammar wallaby blastocysts after embryonic diapause.     

On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

Activation of solubilized liver membrane adenylate cyclase by guanyl nucleotides.

Liver plasma membranes of hypophysectomized rats were purified, treated with 0.1 m Lubrol-PX and centrifuged at 165,000g for 1 h. The detergent solubilized 50% of the membrane protein; adenylate cyclase activity was present in the supernatant fraction. Optimal substrate concentration of the soluble enzyme was 0.32 mm ATP. Basal activity of 25 preparations of the solubilized enzyme ranged from 124 to 39 pmol cyclic AMP/mg protein/10 min. The solubilized enzyme retained the same sensitivity to activation by guanyl nucleotides as was present in the membrane preparation from which it was derived. Relative sensitivity of the solubilized enzyme with 0.1 mm nucleotides or -side was GDP > GTP > GMP > guanosine; GMP-PNP = GMP-PCP > ITP > GTP. GTP, GMP-PCP, GMP-PNP and other nucleotides were hydrolyzed by phosphohydrolases present in liver membranes that were solubilized with Lubrol-PX along with adenylate cyclase. The presence of the ATP regenerating system in the adenylate cyclase assay also aided in maintaining guanyl nucleotide concentrations. The degree of adenylate cyclase activation by guanyl nucleotides was not related to the sparing effects of nucleotides on substrate ATP hydrolysis. These findings demonstrate that activation of adenylate cyclase by nucleotides is a consequence of a nucleotide-enzyme interaction that is independent of membrane integrity.     

MICROASSAY OF ADENOSINE-3′,5′-MONOPHOSPHATE (CYCLIC AMP) IN BRAIN AND OTHER TISSUES BY THE LUCIFERIN-LUCIFERASE SYSTEM1

Abstract— A simple, sensitive and specific method for assaying cyclic AMP in various tissues is reported. Cyclic AMP was isolated from contaminating nucleotides and was converted to ATP with a phosphodiesterase-myokinase-pyruvate kinase system. The ATP was determined enzymically in a liquid scintillation counter by the firefly luciferin-luciferase technique. This procedure was capable of detecting as little as 5 × 10^?14 mol of cyclic AMP and could therefore be used for analyses on less than 1 mg of brain. The assay was reproducible and linear over a wide range of tissue concentrations. In the rat, the highest levels of cyclic AMP (2.7–4.2 pmol/mg wet wt. of tissue) were present in the pineal, heart, pituitary, thyroid, cerebellar cortex, kidney, adrenal, liver and pyloric region of the stomach; intermediate levels (1.5–2.7 pmol/mg wet wt. of tissue) were found in testis, skin, aorta, intestine, submaxillary gland, spleen, muscle and cerebral cortex, moderately low levels (1.0–1.5 pmol/mg wet wt. of tissue) were found in lung, trachea and greater curvature of the stomach; whereas low levels (0.15–0.60 pmol/mg wet wt. of tissue) were found in adipose tissue.     

Continuous-flow bioluminescent dtermination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25°C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.     

Submersible microbial fuel cell sensor for monitoring microbial activity and BOD in groundwater: Focusing on impact of anodic biofilm on sensor applicability

A sensor, based on a submersible microbial fuel cell (SUMFC), was developed for in situ monitoring of microbial activity and biochemical oxygen demand (BOD) in groundwater. Presence or absence of a biofilm on the anode was a decisive factor for the applicability of the sensor. Fresh anode was required for application of the sensor for microbial activity measurement, while biofilm‐colonized anode was needed for utilizing the sensor for BOD content measurement. The current density of SUMFC sensor equipped with a biofilm‐colonized anode showed linear relationship with BOD content, to up to 250 mg/L (～233 ± 1 mA/m²), with a response time of <0.67 h. This sensor could, however, not measure microbial activity, as indicated by the indifferent current produced at varying active microorganisms concentration, which was expressed as microbial adenosine‐triphosphate (ATP) concentration. On the contrary, the current density (0.6 ± 0.1 to 12.4 ± 0.1 mA/m²) of the SUMFC sensor equipped with a fresh anode showed linear relationship, with active microorganism concentrations from 0 to 6.52 nmol‐ATP/L, while no correlation between the current and BOD was observed. It was found that temperature, pH, conductivity, and inorganic solid content were significantly affecting the sensitivity of the sensor. Lastly, the sensor was tested with real contaminated groundwater, where the microbial activity and BOD content could be detected in <3.1 h. The microbial activity and BOD concentration measured by SUMFC sensor fitted well with the one measured by the standard methods, with deviations ranging from 15% to 22% and 6% to 16%, respectively. The SUMFC sensor provides a new way for in situ and quantitative monitoring contaminants content and biological activity during bioremediation process in variety of anoxic aquifers. Biotechnol. Bioeng. 2011;108: 2339–2347. © 2011 Wiley Periodicals, Inc.     

A facile radiometric technique for measuring ATP

A facile radiometric technique for measuring ATP in samples of biologic origin is presented. The d-glucose-6-phosphate produced by the phosphorylation of excess tritiated d-glucose with crystalline hexokinase is stoichiometrically equivalent to the ATP present. Product is selectively precipitated with ethanolic barium acetate, washed with ethanol and the tritium label counted in a scintillation spectrometer. The technique is especially suitable for the measurement of ATP in large numbers of samples (50–100) and offers acceptable sensitivity down to 62 fmoles.     

A direct and sensitive determination of histamine in acid-deproteinized biological samples by high-performance liquid chromatography

A convenient method for the routine measurement of histamine (HA) in biological samples was developed. This method does not require any preliminary purification or concentration of HA, and features high sensitivity, specificity, and reliability. The method consists of the direct application of the acid-deproteinized sample to high-performance liquid chromatography on a sulfonated polystyrene column with detection by means of a postcolumn fluorogenic reaction with o-phthaladehyde. The detection limit was found to be 0.1 pmol (signal-to-noise ratio = 3). The coefficient of variation for measurements of 10 pmol of standard histamine was 1.1%. Each chromatography takes only 10 min and therefore more than 50 samples can be measured in a day. The high sensitivity of the method allows it to be applied even to samples of very low HA concentration such as human plasma without any procedure for concentration of the sample, and further, only 0.1 ml of the sample is necessary for determination. The method was applied to compare the HA levels of the whole blood and plasma of man and various animals. Applications of the method to the supernatant of rat peritoneal mast cell incubates and to extracts of mouse brain and stomach are also described.     

A competitive binding assay for fructose 2,6-bisphosphate

A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P2 to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol protomer) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6-[2-32P]P2 and samples containing varying concentrations of fructose 2,6-P2. The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P2 in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P2, ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. This new assay is simple, direct, rapid, and does not require pretreatment of tissue extracts.     

Elevated plasma vitamin B12 levels and cancer prognosis: A population-based cohort study

BackgroundElevated plasma vitamin B12 levels (cobalamin, Cbl) are associated with increased short-term cancer risk among patients referred for this laboratory measurement. We aimed to assess prognosis in cancer patients with elevated plasma Cbl.MethodsWe conducted a population-based cohort study using data from Danish medical registries during 1998–2014. The study included 25,017 patients with a cancer diagnosis and Cbl levels of 200–600 pmol/L (reference/normal range), 601–800 pmol/L and >800 pmol/L measured up to one year prior to diagnosis, and a comparison cohort of 61,988 cancer patients without a plasma Cbl measurement. Patients treated with Cbl were excluded. Survival probability was assessed using Kaplan–Meier curves. Mortality risk ratios (MRR) were computed using Cox proportional hazard regression, adjusted for age, sex, calendar year, cancer stage and comorbidity, scored using the Charlson comorbidity index.ResultsSurvival probabilities were lower among patients with elevated Cbl levels than among patients with normal levels and among members of the comparison cohort [(1-year survival,%) Cbl: 200–600 pmol/L: 69.3%; 601–800 pmol/L: 49.6%; >800 pmol/L: 35.8%; comparison cohort: 72.6%]. Thirty-day mortality was elevated for patients with Cbl levels of 601–800 pmol/L or >800 pmol/L, compared to patients with levels of 200–600 pmol/L [(MRR (95% confidence interval): 601–800 pmol/L vs. 200–600 pmol/L: 1.9 (1.6–2.2); >800 pmol/L vs. 200–600 pmol/L: 2.7 (2.4–3.1)]. This association remained robust for 31–90-day and 91–365-day mortality, showing similar dose-response patterns.ConclusionCancer patients with elevated Cbl levels had higher mortality than those with normal Cbl levels. These findings may have clinical significance for assessing the prognosis of cancer patients.     

Energy status of nonmatured and in vitro-matured domestic cat oocytes and of different stages of in vitro-produced embryos: enzymatic removal of the zona pellucida increases adenosine triphosphate content and total cell number of blastocysts

In this study, we evaluated the adenosine triphosphate (ATP) content of individual domestic cat oocytes before and after in vitro maturation and of different stages of in vitro-produced embryos. To investigate the effects of assisted-hatching technique on the ATP content and total cell number, the zona pellucida of in vitro-produced blastocysts and expanded blastocysts (recovered 144 h postinsemination [hpi]) was completely removed by pronase treatment. The average (mean +/- SEM) ATP content of nonmatured oocytes (3.47 +/- 0.18 pmol) was significantly (P < 0.01) higher than that of in vitro-matured oocytes (2.17 +/- 0.10 pmol). After in vitro fertilization and culture, the ATP content of two-cell stages (24 hpi) was 1.17 +/- 0.08 pmol, which increased to 1.47 +/- 0.19 and 1.88 +/- 0.32 pmol at the four- (40 hpi) and eight-cell (48 hpi) stages, respectively. The ATP content then decreased to 1.48 +/- 0.10 pmol in 16-cell embryos (64 hpi), reaching a minimum of 0.49 +/- 0.04 pmol at the morula stage (120 hpi). Blastocysts, expanded blastocysts (both 144 hpi), and hatching blastocysts (192 hpi) revealed ATP levels of 1.05 +/- 0.09, 1.79 +/- 0.01, and 4.17 +/- 0.21 pmol, respectively. After enzymatic removal of the zona pellucida (ERZP) at 144 hpi, ATP content and total cell numbers of blastocysts (4.15 +/- 0.37 pmol of ATP, 328.3 +/- 48.5 cells) and expanded blastocysts (5.81 +/- 0.54 pmol of ATP, 430.1 +/- 29.7 cells) analyzed at 192 hpi were significantly (P < 0.001) higher than in their nontreated counterparts (blastocysts: 1.00 +/- 0.09 pmol of ATP, 65.3 +/- 4.6 cells; expanded blastocysts: 1.79 +/- 0.11 pmol of ATP, 121.4 +/- 6.5 cells). Our study describes, to our knowledge for the first time, changes in the energy status of domestic cat oocytes before and after maturation and during in vitro development after fertilization. The ERZP markedly increased the ATP content and total cell number of blastocyst stages, suggesting that this technique may improve the quality and viability of in vitro-produced domestic cat embryos.     

Family physician and endocrinologist coordination as the basis for diabetes care in clinical practice

Background

The role of estrogens in male physiology has become evident. However, clinically useful normative data for estradiol secretion in boys has not previously been established due to the insensitivity of current methods used in clinical routine. By use of a validated ultra-sensitive extraction RIA, our aim was to establish normative data from a group consisting of healthy boys in prepuberty and during pubertal development.

Methods

Sixty-two 24-hours serum profiles (6 samples/24 hours) were obtained from 44 healthy boys (ages; 7.2–18.6 years) during their pubertal development, classified into five stages: prepuberty (testis, 1–2 mL), early (testis, 3–6 mL), mid (testis, 8–12 mL), late-1 (testis,15–25 mL, not reached final height) and late-2 (testis,15–25 mL, reached final height). Serum estradiol was determined by an ultra- sensitive extraction radioimmunoassay with detection limit 4 pmol/L and functional sensitivity 6 pmol/L.

Results

Mean estradiol concentrations during 24-hours secretion increased from prepuberty (median: <4 (5–95 percentiles: <4 – 7) pmol/L) to early puberty (6 (<4 – 12 pmol/L) but then remained relatively constant until a marked increase between mid-puberty (8 (4 – 17) pmol/L) and late-1 (21 (12 – 37) pmol/L) puberty, followed by a slower increase until late-2 puberty (32 (20 – 47) pmol/L). The diurnal rhythm of serum estradiol was non-measurable in pre- and early puberty, but discerned in mid-puberty, and become evident in late pubertal stages with peak values at 0600 to 1000 h.

Conclusion

With the use of an ultra-sensitive extraction RIA, we have provided clinically useful normative data for estradiol secretion in boys.     

Calcium pool size modulates the sensitivity of the ryanodine receptor channel and calcium-dependent ATPase of heavy sarcoplasmic reticulum to extravesicular free calcium concentration

We have examined calcium cycling and associated ATP consumption by isolated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditions believed to exist in resting muscle. Our goals were to estimate the magnitude of calcium cycling under those conditions and identify the main mechanisms involved in its regulation. The integrity of the HSR vesicles was documented by the retention of [¹⁴C]-sucrose and electron microscopy. HSR actively exchanged Ca²⁺ with the medium through a partially open ryanodine-binding channel (RyR), as evidenced by the rapid attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium red (RR) or collapsed by its opening with caffeine. The ATP dependency was evidenced by the sustained ATP consumption after the steady state was attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR vesicles were incubated in a comparatively large pool of calcium (≈1 μmol/mg HSR protein), ATP consumption was 1–1.5 μmol × [min × mg protein]⁻¹ at 0.1 μM free Ca²⁺. Under such conditions, the main regulator of the sarcoplasmic Ca²⁺-dependent ATPase (SERCA) was extravesicular-free Ca²⁺ concentration, with a four- to fivefold increase between 0.1 and 2 μM Ca²⁺, whereas RyR channel activity and the replenishment of the HSR vesicles had only a modest effect on ATP consumption. When calcium pool size was reduced to 0.1 μmol/mg HSR protein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca²⁺ at equilibrium (≈0.07 μM following an initial concentration of 0.1 μM), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large calcium pool, the sensitivity of both RyR channel and SERCA to extravesicular free Ca²⁺ concentration as well as to caffeine and RR was markedly enhanced. Conclusions: (1) In conditions present in resting muscle, HSR calcium is in dynamic equilibrium with the medium through a partially open RyR channel, which requires continuous ATP hydrolysis. (2) The availability of calcium is a major determinant of the sensitivity of both RyR channel and SERCA to free extravesicular Ca²⁺ and possibly other stimuli. (3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fraction of muscle energy demands and further suggest that restricting calcium availability may enhance the energetic demands of this process. J. Cell. Physiol. 175:283–294, 1998. © 1998 Wiley-Liss, Inc.     

Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes

Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at -80 degrees C in groups of 10-20 (GSH) or 5-10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P < 0.05) of GSH (n = 207, 9.82 +/- 0.71 pmol/oocyte; n = 104, 9.73 +/- 0.81 pmol/oocyte; n = 108, 7.89 +/- 0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n = 217, 36.26 +/- 11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n = 70, 0.97 +/- 0.07 pmol/oocyte; TCM-PVA, n = 117, 0.81 +/- 0.13 pmol/oocyte; TCM-MAP, n = 107, 1.02 +/- 0.18 pmol/oocyte; NCSU-pFF, n = 134, 0.71 +/- 0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes.