Effects of albumin, dextran, and hyaluronidase on pulmonary interstitial conductivity

The fluid conductivity of albumin solutions of various concentrations relative to that of saline was measured in the interstitium surrounding a short segment of a large (1.5- to 3-mm-diam) blood vessel of an isolated rabbit lung of which air spaces and vasculature were filled with silicon rubber. At a constant driving pressure, the flow of the following solutions was measured sequentially: normal saline and albumin solution (3, 5.5, 8, or 15 g/100 ml saline), hyaluronidase solution (0.02 g/100 ml), and albumin solution (same concentration used before hyaluronidase solution). The albumin-to-saline flow ratios averaged 1.00 +/- 0.23 (SD), 1.01 +/- 0.21, 1.32 +/- 0.63, and 1.54 +/- 0.36 for albumin concentrations of 3, 5.5, 8, and 15 g/100 ml, respectively. These ratios were higher than the corresponding values of 0.88, 0.78, 0.72, and 0.5 expected if the flow of albumin solution were to depend only on fluid viscosity. The flow of dextran and hyaluronan solutions was more viscosity dependent than the flow of albumin solutions. The increased flow of albumin solution could be the result of a reduced excluded volume of albumin caused by collagen and glycosaminoglycans with an increased albumin concentration. The flow of hyaluronidase solution was 24 +/- 22 (SD)-fold (n = 36) larger than the flow of albumin solution. Thus hyaluronan was responsible for most of the hydraulic resistance of the interstitium to bulk flow. After its degradation, the flow of albumin solution became more viscosity dependent. The interaction between plasma proteins and glycosaminoglycans in the pulmonary interstitium could serve to enhance clearance of microvascular filtrate, particularly under conditions of large protein leaks.     

Blood–Brain Barrier Transport of Valproic Acid

Valproic acid distribution in brain is less than that of other anticonvulsants such as phenytoin or phenobarbital. Possible mechanisms for this decreased distribution space in brain include (a) increased plasma protein binding of valproate relative to the other anticonvulsants and (b) asymmetric blood-brain barrier (BBB) transport of valproate such that the brain-to-blood flux exceeds the blood-to-brain flux. These mechanisms are investigated in the present studies using the intracarotid injection technique in rats and rabbits. In the rat, the brain uptake index (BUI) of [14C]valproate relative to [3H]water is 51 +/- 6%, indicating the blood-to-brain transport of water is twofold greater than that of valproate. However, the BUI of [14C]valproate relative to [3H]water decreased with time after carotid injection during a 4-min washout period, which indicates that brain-to-blood transport of valproate is greater than that of water. This suggests that the permeability of the BBB to valproate is polarized, with antiluminal permeability being much greater than luminal permeability. In rabbits, the BUI of [14C]valproate is 47 +/- 7% in newborns and 17 +/- 6% in adult animals. However, the high drug extraction in newborns may be attributed to decreased cerebral blood flow in the neonate as the BBB permeability-surface area (PS) products are unchanged (e.g., PS = 0.13 and 0.11 ml min-1 X g-1 in the newborn and adult rabbit, respectively). With regard to plasma protein binding effects on valproate transport, brain valproate uptake was also measured in the presence of human, lamb, pig, rat, horse, goat, hamster, dog, and mouse sera. Higher brain uptakes were observed when the unbound fraction of drug increased. However, our data indicate that a fraction of the valproic acid entering the capillaries bound to plasma proteins had the capacity to equilibrate with brain because of enhanced drug dissociation from albumin in the brain microcirculation. Since plasma protein-bound valproate is available for uptake by brain, the major factor underlying the diminished distribution of the drug in brain appears to be the asymmetric transport properties of the BBB to valproic acid.     

Effect of atrial natriuretic factor on plasma vasopressin in conscious rats

An intravenous (IV) bolus injection (10 μg) of synthetic rat atrial natriuretic factor [ANF (Arg 101-Tyr 126)] into normal conscious Sprague-Dawley rats produced a significant decrease of plasma arginine vasopressin (AVP) while 1-, 2-, and 5-μg doses exerted no such effect. Mean arterial blood pressure (MAP) was lowered about 15 mmHg by an IV 10 μg bolus injection of ANF. When plasma AVP rose significantly in rats exposed to such osmotic stimuli as 600 mM NaCl and 900 mM mannitol intraperitoneally (IP), subsequent IV injection of ANF (10 μg) markedly depressed this parameter. Lower doses of ANF were ineffective against 600 mM NaCl IP. The significant elevation of plasma AVP levels by hypertonic sucrose 900 mM IP was not modified by ANF (10 μg). Blood pressure remained unchanged after IP administration of various osmotic stimuli, except mannitol, and in all these experiments an IV bolus of ANF exerted a lowering effect on MAP. Seventy-two hr water deprivation (mixed osmotic and volume stimulus) resulted in elevated plasma AVP levels which were unaffected by an IV bolus injection of ANF at doses of 0.06–10 μg. Immunoreactive ANF (IR-ANF) rose in plasma to 39.3±13 ng/ml 1 min after an IV bolus injection of 10 μg ANF, dropping to 1.01±0.2 ng/ml after 5 min and to 0.32±0.01 ng/ml after 10 min (when ANF and AVP interactions were studied), but still remained approximately six times higher than in control rats. These results suggest that, in the conscious rat, only pharmacological levels of ANF observed after an IV bolus infusion may influence both resting and osmotically-stimulated AVP levels.     

Enhancement of Tryptophan Uptake by Divalent Cations in the Absence of Sodium Ions

Abstract: Nations were found to inhibit the uptake of L-tryptophan into synaptosomes with a shallow dose-response curve. Almost maximal inhibition was obtained with 10 mM-Na⁺. The divalent cations Ca²⁺ and Mg²⁺ were shown to be responsible for the increased uptake of L-tryptophan in the absence of Na⁺ ions. Other divalent cations also promoted tryptophan uptake under this condition (Ca²⁺ < Mg²⁺ < Mn²⁺ < Fe²⁺ < Zn²⁺ < Cu²⁺). It was concluded that monovalent chelate complexes were responsible for this enhancing effect. The measured L-tryptophan uptake was the net product of membrane bound and unbound tryptophan. Both bound and unbound tryptophan were increased in the presence of divalent cations. If no divalent cations were added to the incubation medium, Na⁺ ions decreased the unbound tryptophan but were without effect on bound tryptophan. Under these circumstances D-tryptophan had no effect on binding of the L-isomer and affected the transport of 1.-tryptophan only at very high does (100 x conc. L-tryptophan). These results suggest that I -tryptophan binds to a stereospecific transport carrier located in the synaptosomal membrane and that Na⁺ ions prevent the translocation of this carrier amino acid complex from the outer to the inner site of the neuronal membrane.     

Effects of Albumin, Amino Acids and Clofibrate on the Uptake of Tryptophan by the Rat Brain

Abstract: The influences of total tryptophan concentration, albumin binding and amino acid competition on the rate of tryptophan influx into rat brain were compared using a single-pass injection technique with tritiated water as a freely diffusible reference. Omission of 3% bovine albumin from a bolus containing tryptophan in Krebs–Ringer bicarbonate buffer injected into the carotid artery increased non-albumin bound (free) tryptophan concentration threefold but tryptophan uptake by only 35% and 30% into forebrain and hypothalamus, respectively. However, tryptophan uptake from injected rat plasma was more markedly elevated when free tryptophan concentration was raised. Thus, when free tryptophan was doubled, but total tryptophan unchanged, by in vitro addition of clofibrate to a plasma bolus, uptake was increased by 53% and 28% into forebrain and hypothalamus respectively. When clofibrate was injected in vivo so that plasma total tryptophan concentration was decreased by 45% but neither free tryptophan nor competing amino acid concentrations were altered, then uptake from a bolus of the rat's own plasma was unchanged. Addition of competing amino acids at physiological concentrations to tryptophan in Krebs-Ringer buffer significantly reduced tryptophan influx into both brain regions, but did not increase the effect of albumin binding. The results indicate that tryptophan uptake into rat forebrain is substantially influenced by albumin binding and competition from other amino acids, but that hypothalamic uptake is less influenced by these factors.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. I

1. This paper contains experiments on the influence of acids and alkalies on the osmotic pressure of solutions of crystalline egg albumin and of gelatin, and on the viscosity of solutions of gelatin. 2. It was found in all cases that there is no difference in the effects of HCl, HBr, HNO₃, acetic, mono-, di-, and trichloracetic, succinic, tartaric, citric, and phosphoric acids upon these physical properties when the solutions of the protein with these different acids have the same pH and the same concentration of originally isoelectric protein. 3. It was possible to show that in all the protein-acid salts named the anion in combination with the protein is monovalent. 4. The strong dibasic acid H₂SO₄ forms protein-acid salts with a divalent anion SO₄ and the solutions of protein sulfate have an osmotic pressure and a viscosity of only half or less than that of a protein chloride solution of the same pH and the same concentration of originally isoelectric protein. Oxalic acid behaves essentially like a weak dibasic acid though it seems that a small part of the acid combines with the protein in the form of divalent anions. 5. It was found that the osmotic pressure and viscosity of solutions of Li, Na, K, and NH₄ salts of a protein are the same at the same pH and the same concentration of originally isoelectric protein. 6. Ca(OH)₂ and Ba(OH)₂ form salts with proteins in which the cation is divalent and the osmotic pressure and viscosity of solutions of these two metal proteinates are only one-half or less than half of that of Na proteinate of the same pH and the same concentration of originally isoelectric gelatin. 7. These results exclude the possibility of expressing the effect of different acids and alkalies on the osmotic pressure of solutions of gelatin and egg albumin and on the viscosity of solutions of gelatin in the form of ion series. The different results of former workers were probably chiefly due to the fact that the effects of acids and alkalies on these proteins were compared for the same quantity of acid and alkali instead of for the same pH.     

Chondroitin sulfate reduces the friction coefficient of articular cartilage

The objective of this study was to investigate the effect of chondroitin sulfate (CS)-C on the frictional response of bovine articular cartilage. The main hypothesis is that CS decreases the friction coefficient of articular cartilage. Corollary hypotheses are that viscosity and osmotic pressure are not the mechanisms that mediate the reduction in the friction coefficient by CS. In Experiment 1, bovine articular cartilage samples (n=29) were tested in either phosphate buffered saline (PBS) or in PBS containing 100mg/ml of CS following 48h incubation in PBS or in PBS+100mg/ml CS (control specimens were not subjected to any incubation). In Experiment 2, samples (n=23) were tested in four different solutions: PBS, PBS+100mg/ml CS, and PBS+polyethylene glycol (PEG) (133 or 170mg/ml). In Experiment 3, samples (n=18) were tested in three solutions of CS (0, 10 and 100mg/ml). Frictional tests (cartilage-on-glass) were performed under constant stress (0.5MPa) for 3600s and the time-dependent friction coefficient was measured. Samples incubated or tested in a 100mg/ml CS solution exhibited a significantly lower equilibrium friction coefficient than the respective PBS control. PEG solutions delayed the rise in the friction coefficient relative to the PBS control, but did not reduce the equilibrium value. Testing in PBS+10mg/ml of CS did not cause any significant decrease in the friction coefficient. In conclusion, CS at a concentration of 100mg/ml significantly reduces the friction coefficient of bovine articular cartilage and this mechanism is neither mediated by viscosity nor osmolarity. These results suggest that direct injection of CS into the joint may provide beneficial tribological effects.     

Effect of active immunisation against testosterone-3-BSA on circulating levels of testosterone, LH, prolactin and testosterone antibody titre in the male rat

Male Sprague-Dawley rats were actively immunised against testosterone-3-bovine serum albumin (T-3-BSA) and on appearance of detectable anti-testosterone antibodies, elevated serum testosterone and LH concentrations were observed. These concentrations reached values of >28 μg/100ml testosterone and 16 μg/100ml LH in some animals after 5 months of immunisation. The corresponding prolactin values did not appear to differ significantly from controls. The circulating bound testosterone fraction as determined by equilibrium dialysis, rose from 65.0 ± 2.75% before immunisation to 98.7 ± 0.75% in those animals possessing high titre antisera. This entailed a nett $\text{decrease}$ in the concentration of unbound steroid from 144 ± 49 ng/100 ml to 78 ± 25 ng/100ml.     

The modification of the lone tryptophan residue in human serum albumin by 2-hydroxy-5-nitrobenzyl bromide. Characterization of the modified protein and the binding of L-tryptophan and benzodiazepines to the tryptophan-modified albumin.

The possible function of the lone tryptophan residue of human serum albumin in the stereospecific binding site for indole and benzodiazepine compounds was investigated by chemical modification. This residue can be selectively modified with 2-hydroxy-5-nitrobenzyl bromide. The modification alters the conformation of the albumin only slightly, as revealed by circular dichroism, fluorescence, and ultraviolet absorption measurements. A decrease in the association constants of L-tryptophan and diazepam of about 30 - 50% and a decrease in the extrinsic Cotton effects of four benzodiazepine derivatives of about 10 - 15% were found as specific effects of the tryptophan modification. The tryptophan modification itself did not change the number of binding sites of diazepam and L-tryptophan. It is suggested that the lone tryptophan residue of human serum albumin is not directly involved in the specific binding site for indole and benzodiazepine compounds. However, the modification alters the properties of the binding site either by an incomplete refolding of the albumin after urea treatment, or a more selective allosteric effect of the modified tryptophan residue.     

Kinetics of bilirubin oxidase and modeling of an immobilized bilirubin oxidase reactor for bilirubin detoxification

The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin-serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin-serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K(1) = 6 x 10(7)M(-1) and K(2) = 4.5 x 10(6)M(-1), respectively. The rate of total bilirubin degradation in bilirubin-serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8-mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350muM and a serum albumin concentration of 500muM.     

Permeability of the Blood-Brain Barrier to the Immunosuppressive Cyclic Peptide Cyclosporin A

Uptake of the immunosuppressive lipophilic peptide cyclosporin A has been measured by a number of techniques. The brain uptake index (BUI) technique in the rat yields only a small BUI value that is not significantly different from that of sucrose and mannitol and is comparable to other published BUI values for this compound. Brain perfusion studies in the guinea pig produce a unidirectional cerebrovascular permeability constant (Kin) of 1.2 +/- 0.28 microliter g-1 min-1 for the hippocampus. Intravenous bolus injection techniques also in the guinea pig characteristically produce a larger Kin value of 2.53 +/- 0.38 microliter g-1 min-1 for the same brain region, even after a correction for the inulin space of the tissue has been made. Apparent penetration of cyclosporin A into the cerebrospinal fluid (CSF) determined with the intravenous bolus injection technique is small with a Kin of 0.79 +/- 0.07 microliter g-1 min-1. However it is suggested that the radioactivity present in CSF is largely tritiated water. Studies with cultured cerebral endothelial cells from the rat have also been carried out and show that the cultured cells take up and accumulate cyclosporin A in vitro, achieving a tissue-to-medium ratio of 20 after 25 min of incubation. It is suggested that cyclosporin A is primarily taken up from lipoprotein at the blood-brain interface but, because of tight junctions at the blood-brain and blood-CSF barriers, becomes effectively trapped in the cerebral endothelial cells and the choroid plexus.     

Palmitic acid incorporation into intramuscular acylglycerols depends on both total and unbound to albumin palmitic acid concentration.

Palmitic acid incorporation into the intramuscular acylglycerols in rat skeletal muscles of different fiber types was investigated at various total and unbound to albumin concentrations by means of the hind-limb perfusion technique. It was found that at simultaneously increasing total and unbound to albumin palmitic acid concentrations in the perfusion medium the incorporation of palmitic acid into acylglycerols increased. However, when the concentration of palmitic acid not bound to albumin was kept constant and the total palmitic acid concentration was increased, the incorporation also increased although markedly less than under former conditions. The increase was most apparent in the muscles composed of slow-twitch oxidative and fast-twitch oxidative-glycolytic fibers where fatty acid uptake is the greatest. These findings suggest that fatty acid incorporation into intramuscular acylglycerols depends not only on the unbound to albumin fatty acid concentration but also, to some extent, on the total fatty acid concentration.     

Pulmonary microvascular response to LTB4: effects of perfusate composition

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.     

Brain uptake of pipecolic acid,amino acids,and amines following intracarotid injection in the mouse

The uptake of pipecolic acid by the mouse brain was compared to that of several amino acids and amines, following an injection of a double-labeled mixture into the carotid artery. In general, BUI (brain uptake index) values were lower in the mouse than those previously reported in the rat. The only exception was proline. Lysine, a precursor of pipecolic acid biosynthesis in brain, showed a higher BUI than pipecolic acid. The BUI ofD,l-[³H]pipecolic acid was found to be 3.39 (at 0.114 mM). This was saturable between a concentration of 0.114 and 3.44 mM. Kinetic analysis suggests the presence of two kinds of transport systems. Substances structurally related to pipecolic acid, such as nipecotic acid, isonipecotic acid,l-proline, and piperidine show a significant inhibitory effect. Among the amino acids tested, only GABA showed an inhibitory effect. Data are reported which, when considered with other findings (5), present evidence that pipecolic acid is (1) synthesized both in vitro and in vivo in the mouse brain, (2) actively transported in vivo into the brain, and (3) taken up in vitro by synaptosomal preparations.     

Effect of salt loading on the body water compartments in rats of different ages

In acute experiments on anaesthetized rats from three age groups (15-20, 25-30 days of postnatal life, adult ones), studies have been made on water basins of the organism after peroral injection of hypertonic (2.5-5%) solutions of NaCl (5 and 10 ml per 100 g of the body weight). It was demonstrated that during ontogenesis, total content of water decreases mainly at the expense of extracellular fluid. Infusion of saline solutions into the stomach of rats decreases fluid content in all water basins, especially in the interstitial one. The level of changes depends on the volume of the injected solution and, to a greater extent, on the concentration of the latter and the age of animals. The described response is due to osmotic transport of water into the alimentary tract, as suggested by the decrease of water content in this tract. The role of the digestive tract in osmotic and volume regulation during peroral salt loading is discussed.     

A highly reactive tyrosine residue as part of the indole and benzodiazepine binding site of human serum albumin.

The interaction of L-tryptophan and four benzodiazepine derivatives with tyrosine-modified human serum albumin was investigated by equilibrium dialysis and circular dichroism measurements. Out of the 18 tyrosine residues of the human serum albumin molecule, only 9 could be modified with tetranitromethane. At least up to a degree of modification of 5, the conformation of human serum albumin was not changed and no crosslinking and fractionation has been found, as revealed from circular dichroism measurements in the far ultraviolet range and from SDS polyacrylamide electrophoresis. The modification of only 2 out of the 9 accessible tyrosine residues of human serum albumin strongly affects the binding of L-tryptophan and diazepam to their common, stereospecific bindining site. This was evidently shown by a reduction of the association constants by more than 90% and by a large reduction of the extrinsic Cotton effects of four benzodiazepines bound to human serum albumin. The numbers of binding sites remained unchanged. Strong evidence was presented that only one tyrosine residue, which reacts faster with tetranitromethane than all others, is mainly involved in the specific indole and benzodiazepine binding site of human serum albumin. The location of this highly reactive tyrosine residue and that of the specific indole and benzodiazepine binding site within the human serum albumin primary structure is discussed.     

Residence time distribution of diazepam in the isolated perfused rat liver. Analysis with the axial dispersion model.

The application of the axial dispersion model to diazepam hepatic elimination was evaluated using data obtained for impulse-response experiments with diazepam in the single-pass isolated perfused rat liver preparation. The transient form of the two-compartment dispersion model was applied to the output concentration versus time profile of diazepam after bolus input of a radiolabelled tracer into the hepatic portal vein (n = 4), providing DN and CLint estimates of 0.251 +/- 0.093 and 135 +/- 59 ml min-1, respectively. In contrast, the one-compartment form of the axial dispersion model, which assumes instantaneous transversal distribution of substance to the accessible spaces within the liver, could not adequately describe the residence time distribution (RTD) of diazepam. Furthermore, the magnitude of DN, a stochastic parameter which characterizes the axial spreading of solutes during transit through the liver, is similar to that determined for non-eliminated substances such as erythrocytes, albumin, sucrose and water. These findings suggest that the dispersion of diazepam in the perfused rat liver is determined primarily by the architecture of the hepatic microvasculature.     

Effect of nonenzymatic glycation of albumin and superoxide dismutase by glucuronic acid and suprofen acyl glucuronide on their functions in vitro.

Acyl glucuronides bind irreversibly to plasma proteins, and one mechanism proposed for this covalent binding is similar to that for glycation of protein by reducing sugars. Because glycation of protein by glucose and other reducing sugars can alter protein function, this lead to the hypothesis that the glycation of proteins by acyl glucuronides may cause similar effects. When human serum albumin (HSA) was incubated with 0.5 M glucose for 5 days, the unbound fractions of diazepam and warfarin were increased by 41 and 35%, respectively, less than that caused by glucuronic acid which increased the unbound fractions by 90% for diazepam and 420% for warfarin. When HSA was incubated with suprofen glucuronide (SG) at a much lower concentration of 0.005 M for only 24 h, the effects on the unbound fractions of diazepam and warfarin to HSA were altered dramatically with increases of 340 and 230%, respectively. After incubation of superoxide dismutase (SOD) with 0.5 or 1 M reducing sugars for 14 days, the enzyme activity decreased to 82 and 61% of initial levels at day 14, respectively, whereas glucuronic acid almost completely inactivated the enzyme activity over the same period. Even at a very low concentration (0.005 M) of SG, SOD activity was reduced significantly to 11% of initial levels by day 14, which was comparable to the effect by 0.5 and 1.0 M concentrations of glucuronic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix associated laser desorption/ionization time of flight mass spectrometry indicated that several equivalents of reducing sugars or SG became attached to albumin after incubation. These results suggest that acyl glucuronides may affect the function of proteins by the formation of glycated protein in vivo and may be associated with the toxicity of xenobiotics metabolized to labile acyl glucuronides.     

Analysis of picogram quantities of protein in subnanoliter-size samples

The ability to measure protein concentration in subnanoliter volumes would be helpful in many biological studies. A microassay for measuring nanogram protein quantities in nanoliter-size samples and an ultramicroassay for measuring picogram quantities in picoliter samples were developed to measure lymphatic protein concentration. Aliquots of a sample solution were mixed with an o-phthalaldehyde mercaptoethanol reagent using micropipetting techniques. Reaction product fluorescence was measured using a modified Leitz MPV-1 microscope as a microfluorometer. Fluorescence varied linearly with albumin concentrations between 1 and 8 g/100 ml. A typical microassay measuring albumin standards at 0.0, 1.0, 2.0, and 4.0 g/100 ml yielded a linear regression of y = 207x + 60 (r = 0.99). Minimum detectable protein concentration was 0.125 g/100 ml. The SE for the albumin standards varied from 0.02 to 0.17 g/100 ml. An ultramicroassay measuring similar standards yielded a linear regression of y = 1180x + 109 (r = 0.96). Minimum detectable protein concentration was 0.028 g/100 ml. The SE for the standards varied from 0.01 to 0.32 g/100 ml.     

Palmitic acid uptake by the rat soleus muscle in vitro.

Abstract: The rate of fatty acid uptake, oxidation, and deposition in skeletal muscles in relation to total and unbound to albumin fatty acids concentration in the medium were investigated in the incubated rat soleus muscle. An immunohistochemical technique was applied to demonstrate whether the albumin-bound fatty acid complex from the medium penetrates well within all areas of the muscle strips. It was found that the percentage of incorporation of palmitic acid into intramuscular lipids was fairly constant, independently of the fatty acid concentration in the medium, and amounted to 63-72% for triacylglycerols, 7-12% for diacylglycerols-monoacylglycerols, and 19-26% for phospholipids. Both palmitic acid incorporation into the muscle triacylglycerol stores and its oxidation to CO2 closely correlated with an increase in both total and unbound to albumin fatty acid concentrations in the incubation medium. Under conditions of increased total but constant unbound to albumin palmitic acid concentrations, the incorporation of palmitic acid into triacylglycerols and its oxidation to CO2 were also increased, but to a lower extent. This supports the hypothesis that the cellular fatty acid metabolism depends not only on the availability of fatty acids unbound to albumin, but also on the availability of fatty acids complexed to albumin.