Oxygen dependence of respiration in rat spinotrapezius muscle in situ

The oxygen dependence of respiration in striated muscle in situ was studied by measuring the rate of decrease of interstitial Po2 [oxygen disappearance curve (ODC)] following rapid arrest of blood flow by pneumatic tissue compression, which ejected red blood cells from the muscle vessels and made the ODC independent from oxygen bound to hemoglobin. After the contribution of photo-consumption of oxygen by the method was evaluated and accounted for, the corrected ODCs were converted into the Po2 dependence of oxygen consumption, V̇o2, proportional to the rate of Po2 decrease. Fitting equations obtained from a model of heterogeneous intracellular Po2 were applied to recover the parameters describing respiration in muscle fibers, with a predicted sigmoidal shape for the dependence of V̇o2 on Po2. This curve consists of two regions connected by the point for critical Po2 of the cell (i.e., Po2 at the sarcolemma when the center of the cell becomes anoxic). The critical Po2 was below the Po2 for half-maximal respiratory rate ( P 50) for the cells. In six muscles at rest, the rate of oxygen consumption was 139 ± 6 nl O2/cm3·s and mitochondrial P 50 was k = 10.5 ± 0.8 mmHg. The range of Po2 values inside the muscle fibers was found to be 4–5 mmHg at the critical Po2. The oxygen dependence of respiration can be studied in thin muscles under different experimental conditions. In resting muscle, the critical Po2 was substantially lower than the interstitial Po2 of 53 ± 2 mmHg, a finding that indicates that V̇o2 under this circumstance is independent of oxygen supply and is discordant with the conventional hypothesis of metabolic regulation of the oxygen supply to tissue.

Phosphorescence quenching microrespirometry of skeletal muscle in situ

We have developed an optical method for the evaluation of the oxygen consumption (V̇o2) in microscopic volumes of spinotrapezius muscle. Using phosphorescence quenching microscopy (PQM) for the measurement of interstitial Po2, together with rapid pneumatic compression of the organ, we recorded the oxygen disappearance curve (ODC) in the muscle of the anesthetized rats. A 0.6-mm diameter area in the tissue, preloaded with the phosphorescent oxygen probe, was excited once a second by a 532-nm Q-switched laser with pulse duration of 15 ns. Each of the evoked phosphorescence decays was analyzed to obtain a sequence of Po2 values that constituted the ODC. Following flow arrest and tissue compression, the interstitial Po2 decreased rapidly and the initial slope of the ODC was used to calculate the V̇o2. Special analysis of instrumental factors affecting the ODC was performed, and the resulting model was used for evaluation of V̇o2. The calculation was based on the observation of only a small amount of residual blood in the tissue after compression. The contribution of oxygen photoconsumption by PQM and oxygen inflow from external sources was evaluated in specially designed tests. The average oxygen consumption of the rat spinotrapezius muscle was V̇o2 = 123.4 ± 13.4 (SE) nl O2/cm3·s ( N = 38, within 6 muscles) at a baseline interstitial Po2 of 50.8 ± 2.9 mmHg. This technique has opened the opportunity for monitoring respiration rates in microscopic volumes of functioning skeletal muscle.

The importance of the unsuppressed glands in the study of intact parathyroid hormone disappearance after parathyroid adenomectomy

BACKGROUND: In the usual techniques for intraoperative intact parathyroid hormone (iPTH) monitoring for primary hyperparathyroidism, the normal glands are implicitly considered suppressed. On the contrary, we believe, as do other researchers, that they are not totally suppressed. METHODS: For this reason, we considered the introduction of an infusion from the unsuppressed normal glands (UNG), described by an influx constant (IC (pg/ml per min)), into the formulation of a two-compartment model. For the blood compartment, we have: C(t)=A.exp(-at)+B.exp(-bt)+EV, where A+B+EV=iPTH concentration at zero time (clamping), EV (equilibrium value)=IC/k, 'a' and 'b' are reciprocals of the time constants of the two exponentials and k=rate constant of elimination from the blood. The experimental data were obtained using an IRMA standard method, collecting samples in 20 patients, during and following adenomectomy. RESULTS: In spite of the variability among the patients, all fits were very good, thus confirming the importance of the UNG contribution to the shaping of the disappearance curve. For this reason, the relationship between the constant infusion from the UNG and the basal iPTH level at the induction of anaesthesia (BV), was studied. CONCLUSIONS: The existence of a negative correlation, together with the determination of a regression curve (IC=6.5BV), not only confirmed our assumptions, but also revealed the theoretical possibility of a priori knowledge of the iPTH contribution from the UNG. Hence, there is a theoretical possibility of discriminating between this contribution and that of the remaining (if any) affected gland(s).

Hepatic removal of two fractions of indocyanine green after bolus injection in anesthetized pigs

In the anesthetized pig, we studied the kinetics after intravenous bolus injection of two fractions of indocyanine green (ICG): the genuine ICGg (95-99% of total) and a degradation product, ICGdp (1-5%). Plasma concentrations were followed in the carotid artery and a hepatic vein. ICGg disappearance curves (n = 7) were biexponential with rate constants alpha = 0.189 +/- 0.021 min-1 and beta = 0.0356 +/- 0.0061 min-1. The hepatic extraction fraction was constant with time. A detailed mathematical analysis showed this to be in disagreement with the conventional assumption that the biexponential plasma disappearance curve is a result of backflux from the liver storage to plasma. In contrast, our observations were predicted by an alternative model assuming temporary extrahepatic, extravasal redistribution during first-order, one-way hepatic uptake. Nevertheless, when a large bolus of sulfobromophthalein (BSP) was injected 20 min after ICG, a net backflux of ICG could be demonstrated, presumably due to countertransport. Thus a sufficient description of ICGg kinetics must include the complex kinetic behavior of the hepatic membrane carrier involved. Mass spectrometry suggested that ICGdp is formed by two ICGg molecules. Plasma elimination of ICGdp was slower (alpha = 0.0094 +/- 0.0007 min-1). Analysis of the bile after bolus injection (n = 2) of ICGdp revealed two possible metabolites of ICGdp that were not found in urine. Since BSP injection did not alter the ICGdp disappearance curve, ICGdp is probably not taken up by the same hepatic membrane carrier as ICGg.

Fate of circulating amino-terminal propeptide of type III procollagen in conscious pigs

The amino-terminal propeptide of type III procollagen (PIIINP, M(r) 42,000) is a promising marker for the formation of type III collagen of granulation tissue in experimental and clinical studies. The disposal kinetics of circulating PIIINP is, however, almost unknown. In conscious pigs with a thoracic duct-venous shunt, 125I-labeled PIIINP was injected intravenously. The initial distribution volume was 2.2 liters, which was 1.7 times the plasma volume (P < 0.01). The disappearance curve was three-phased, with an initial steep decline (t1/2 58 min), followed by two slower phases (t1/2 239 min and 289 h). Consecutive gel filtrations showed that the initial slope of the plasma disappearance curve corresponded to the plasma clearance of the intact PIIINP. The initial plasma clearance was 26.5 ml plasma/min, whereas the urinary clearance was 8.7 ml plasma/min (P < 0.01). The other components of the plasma disappearance curve originated from the formation and disappearance of a high and a low molecular weight (MW) fraction as part of the degradation of PIIINP. The high MW fraction (approximately M(r) 90,000) was similar to a previously described, but not further characterized, PIIINP immunoreactive component. The existence of the low MW fraction (approximately M(r) 20,000) has not been reported before. The lymphatic recirculation of intact PIIINP was rapid, and the lymph-serum ratio was almost constant within 1 h of injection. We conclude that the t1/2 of circulating PIIINP is 58 min, that PIIINP escapes the circulation very quickly, and that the degradation of PIIINP includes at least two intermediary steps.

Disposal and distribution of rT3 in humans: a new double-tracer kinetic study

A satisfactory definition of reverse 3,3',5'-triiodothyronine (rT3) kinetics in humans cannot be obtained if the plasma disappearance curve of the injected labeled hormone is the only experimental data available; most of the kinetic parameters can only be bounded within ranges showing unacceptable variabilities. To gain additional experimental data a double-tracer approach is proposed. After simultaneous injection of [125I]rT3 and 131I the following three experimental curves were determined in plasma: 1) the disappearance of [125I]rT3, 2) the disappearance of 131I, and 3) the appearance of 125I generated in vivo from labeled rT3 degradation. Combined analysis of these three curves, based on a complex six-compartment model, was developed and applied to data obtained in a group of normal subjects. Through this new analysis, fractional disposal rates and fractional exchange rates between the plasma compartment and the periphery are uniquely determined. The main physiologically interesting information on the degradation of the hormone that emerges from these studies are 1) all degradative pathways of rT3 generate iodide, being in all cases the [125I]rT3 dose completely recovered as 125I in plasma; and 2) most rT3 is degraded (65–90%) in peripheral tissues rapidly exchanging with the plasma pool. The extended experimental base is not yet sufficient to compute unique values for production rate (PR) and body mass (Qt); the validity of estimates of PR and Qt is based on the assumption that injected [125I]rT3 is able to trace all rT3 peripherally produced (from thyroxine). The new approach yields ranges for PR and Qt (1.12–2.15 micrograms/h and 2.88–8.24 micrograms) much narrower than those computable from the [125I]rT3 disappearance curve only (1.12–5.07 micrograms/h for PR and 2.88-23.7 micrograms for Qt).

Disappearance of palmitic acid from plasma of fetal and newborn sheep

These studies were undertaken to measure the kinetic constants that characterize the disappearance of a representative free fatty acid (FFA) from the plasma of fetal and newborn sheep. A bolus of albumin-complexed [14C]palmitic acid was infused intravenously, and during the next 8 min, 24 arterial samples were collected to characterize the disappearance curve. Palmitic acid disappearance from plasma was well described by a double-exponential model. When birth was simulated in utero, kinetic values were not changed by cooling. However, after intrauterine ventilation with O2, the volume of distribution of the FFA increased 29%, its plasma clearance rate decreased 26%, and its apparent half-life in the plasma lengthened from 0.8 to 1.2 min (all P < 0.01, n = 8). After umbilical cord occlusion, plasma clearance rate decreased a further 19% and half-life lengthened to 1.4 min. About 60% of the increase in FFA concentration during simulated birth is explained by increased release from adipose stores, and 40% is explained by decreased clearance. Further experiments tested the influence of FFA concentrations themselves. After infusion of unlabeled FFA, clearance of the tracer decreased 23% (P < 0.05, n = 5), a result consistent with a saturable membrane transporter of FFAs.

Clinical pharmacology of a novel diarylsulfonylurea anticancer agent.

LY186641 (diarylsulfonylurea, [DSU]) is a novel anticancer agent because of its unique diarylsulfonylurea chemical structure, broad-spectrum antisolid-tumor activity in preclinical models, presumed novel mechanism of action and preclinical toxicities of methemoglobinemia (Met Hgb) and decreased red blood cell (RBC) survival. In this study, the in vitro drug sensitivity of human tumors as well as clinical pharmacology and toxicology of DSU in patients with cancer were examined. DSU was administered orally, daily for 7 days with a 3-week treatment cycle. Dose-limiting toxicities were Met Hgb and RBC hemolysis. The maximum-tolerated dose was found to be 1,200 mg/m2/d for 7 days. In pharmacokinetic studies, DSU was found to have a prolonged serum half-life (approximately 30 hours) and a large area under the plasma disappearance curve (8,883.3 micrograms.hr/mL at 1,200 mg/m2/d). A partial remission was observed in one patient with refractory ovarian cancer. In conclusion, DSU can be safely administered to cancer patients and does display antitumor activity. Potential means of obviating the toxicities of this compound are discussed.

Evidence for second metabolic pathway for O2 from PtiO2 measurements in denervated cat carotid body

O2 microelectrode studies were conducted in the cat carotid body (CB) to investigate the hypothesis that there is a second, low affinity metabolic pathway for O2 in addition to classical oxidative metabolism. Tissue PO2 (PtiO2) and O2 disappearance rates (dPO2/dt) after brief blood flow occlusion were measured with recessed cathode microelectrodes (tip diameter less than 5 microns) at 150 sites in 15 normal cats (controls) and at 154 sites in 5 cats in which one CB had been denervated 2 or 3 days before the experiments. Mean PtiO2 was slightly higher in denervated CBs: 79.6 +/- 1.6 (SE) Torr compared with 76.4 +/- 2.0 Torr for controls (P = not significant). Mean dPO2/dt was 8.4% faster: -8.42 +/- 0.28 Torr/s compared with -7.77 +/- 0.43 Torr/s for controls (P less than 0.05). The O2 consumption rate (VO2), calculated from dPO2/dt correcting for cat oxyhemoglobin, was 7.5% higher: 1.62 and 1.51 ml.100 g-1.min-1, respectively, for denervated and control CBs (P less than 0.05). The apparent Michaelis-Menten constant, Kmapp (defined as the PtiO2 where dPO2/dt decreased by 50% from the initial rate during the first 3 s after occlusion) was determined for each O2 disappearance curve. After denervation, Kmapp decreased significantly by -47%: 12.0 +/- 1.3 Torr compared with 22.6 +/- 2.5 Torr for controls (P less than 0.01). The data provide evidence for a second metabolic pathway for O2 in the CB that loses its influence on VO2 after denervation.