Mechanisms of hepatic disposition of polystyrene microspheres in rats: effects of serum depend on the sizes of microspheres.

To study the mechanisms of the hepatic disposition of polystyrene microspheres (MS), effects of serum on their hepatic disposition characteristics were investigated for MSs with particle sizes of 50 nm (MS-50) and 500 nm (MS-500) by isolated liver perfusion experiments. It was revealed that serum in the perfusate inhibited and promoted the hepatic disposition of MS-50 and MS-500 at 37 degrees C, respectively. However, pre-heating at 56 degrees C or pre-treatment with anti-C3 antibody of serum reduced the promotive effect of serum on the hepatic uptake of MS-500, suggesting that the complement system should be involved as opsonins for the hepatic uptake of MS-500. Hepatic disposition of both MSs at 4 degrees C was reduced by the addition of serum into the perfusate, which could be ascribed to the reduction of the surface hydrophobicity of MSs due to the adsorption of serum proteins onto the surface of MSs and to resultant decrease in non-specific disposition to the liver. From these results, serum was found to function both as the opsonin to enhance the hepatic uptake of MSs and as the inhibitor by reducing non-specific interaction between MSs and the plasma membrane. Whether serum promotes or inhibits the hepatic disposition of MSs would be dependent on the particle sizes of MSs.     

Human serum amyloid A. Three hepatic mRNAs and the corresponding proteins in one person.

Serum amyloid A protein (SAA) is a major acute-phase protein in humans and most other mammals. In addition, it is the serum precursor of the major protein constituent of reactive amyloid fibrils. Sequence analyses have identified a number of polymorphic forms of human SAA and amyloid A protein (AA), but the question of the number of genes encoding SAA in the human has not been addressed. In addition, there are insufficient data to predict whether one form of SAA predisposes to amyloid fibril formation. In the present study three separate SAA proteins have been isolated from the plasma of one individual and completely sequenced. While two of the SAA forms (SAA2 alpha and SAA2 beta) differ from each other only at position 71, they differ from the most abundant form (SAA1) at seven and eight other positions, respectively. Nucleotide sequencing of cDNAs from a liver library of this individual identified all three mRNs coding for these proteins and proved that: (a) the often-reported absence of arginine at the amino terminus of SAA proteins must result from proteolytic processing of the protein; (b) the polymorphism involving histidine and arginine at position 71 is present at the DNA level and therefore is not due to an event at the translational level; (c) there are at least two genes coding for human SAA. Comparison of these data to published sequences of SAA and AA proteins may help in identifying genetically determined forms of SAA which predispose to reactive amyloid fibril formation.     

The role of serum proteins in acid-base equilibria

Serum proteins act as weak acids and participate in acid-base balance. Their effects are imprecisely quantified; in particular, the roles of albumin and globulins need reevaluation. We approached the problem in three steps. First, in artificial solutions resembling serum but with human serum albumin as the only protein moiety, we varied the strong ion difference (SID), partial pressure of carbon dioxide (Pco2) and the concentration of albumin [( Alb]) and fixed the concentration of inorganic phosphate [( Pi]). We measured pH and derived the charges on albumin. Second, extending the work of Stewart (Stewart PA. How to understand acid-base. A quantitative acid-base primer for biology and medicine. New York: Elsevier, 1981:1-286), we developed a mathematical model that solves for pH and for the charges on albumin as functions of SID, Pco2, [Pi], and [Alb]. The calculated values fit the observed values well; that is, the model describes well the behavior of these solutions over a wide range of simulated complex acid-base disturbances. Finally, in human serum samples containing both albumin and globulins, we varied SID, Pco2, and total protein concentration [( TP]); we fixed [Pi] and then measured pH and derived the charges on proteins as above. When we applied to these data the computer model developed for albumin alone, the calculated pH and derived charges on albumin values agreed well with the observed pH and derived charges on proteins. We conclude first that human serum globulins play a negligible role in acid-base equilibria, and second, that in normal human serum at pH 7.40 with [TP] = 7 and [Alb] = 4.3 gm/dl, the charges attributed to proteins are approximately 12 mEq/L; this is substantially less than the value of approximately 17 mEq/L given by many contemporary texts, based on work of van Slyke et al. (van Slyke DD, Hastings AB, Hiller A, Sendroy J Jr. Studies of gas and electrolyte equilibria in blood. XIV. Amounts of alkali bound by serum albumin and globulin. J Biol Chem 1928;79:769-80). These findings should be considered when evaluating acid-base balance in patients with abnormal serum albumin concentration, for example, when interpreting values of the anion gap.     

Role of SLC xenobiotic transporters and their regulatory mechanisms PDZ proteins in drug delivery and disposition.

Various types of xenobiotic (or drug) transporters have been recently identified to play important roles as barriers against toxic compounds and influx pumps to take up nutrients into the body. Since those xenobiotic transporters generally have wide range of recognition specificity and accept various types of compounds as substrates, localization and functional expression of such transporters could be one of the critical factors that affect the disposition and subsequent biological activity of therapeutic agents. Identification and characterization of drug transporters have given us a scientific basis for understanding drug delivery and disposition, as well as the molecular mechanisms of drug interaction and inter-individual/inter-species differences. To precisely understand pharmacological roles of the transporters in the body, it is also important to clarify molecular mechanisms involved in regulation of the transporters. As a first step to clarify the regulatory mechanisms that govern cell-surface expression and/or function of these transporters, recent researches have focused on PDZ (PSD-95/Discs-large/ZO-1) binding motif localized on carboxylic terminus of several types of xenobiotic transporters. Most of the transporters showing direct interaction potential with the PDZ domain-containing proteins are expressed on apical membranes in epithelial cells of kidney and/or small intestine, implying that such protein-protein interaction may play a role in apical localization of the transporters. In this mini-review article, we summarize importance of transporters and their regulatory mechanisms in drug delivery and disposition, focusing on several aspects of transporter-mediated drug targeting.     

Narcotic effects on hepatic disposition of sulfobromophthalein in rats

Ascending morphine doses above 5 mg/kg s.c. progressively reduced plasma clearance of sulfobromophthalein (BSP) and raised hepatic levels of this dye in rats. The narcotic reduced the elimination constant of BSP without affecting its volume of distribution. Because abdominal surgery markedly reduced plasma clearance of BSP, no further effect of morphine could be shown in rats with bile cannulas. In duct-cannulated animals morphine had no effect on BSP concentration in bile, but did raise hepatic BSP levels while reducing bile flow and biliary BSP content. The narcotic also lowered the biliary transport maximum of BSP. The effects on BSP disposition were demonstrated acutely after morphine administration but had subsided completely by 1 and 2 days after giving narcotic. The present findings suggest that morphine impaired the secretion of BSP into bile by a mechanism not involving biliary occlusion and thereby enhanced retention of this dye in liver and plasma.     

Opioid effects on hepatic disposition of dyes in mice

Morphine administration acutely reduced plasma clearance of sulfobromophthalein (BSP) in mice and increased hepatic retention of this dye. Increasing morphine doses from 5 to 40 mg/kg s.c. progressively raised plasma and liver BSP levels. Morphine-treated mice, warmed to reverse hypothermia, still had higher plasma and liver BSP levels. The narcotic also raised plasma levels of two dyes which are not conjugated, indocyanine green and dibromosulfophthalein. Naloxone reversed morphine-induced elevation of plasma BSP levels. In bile duct-ligated mice, plasma BSP levels were very high but hepatic BSP levels remained low, both after saline or morphine. Thus, the effects of morphine on BSP disposition differed from those of biliary occlusion. BSP content in bile was reduced by morphine, as dye levels were raised in plasma and hepatic parenchyma. In bile duct-cannulated mice morphine increased BSP levels in plasma and liver whereas reducing the amount of dye eliminated in bile.     

Surface hydrophobicity of particles is not necessarily the most important determinant in their in vivo disposition after intravenous administration in rats.

The in vivo disposition of polystyrene microsphere (MS) with the particle size of 50 nm (MS-50) and lecithin-coated MS-50 (LMS-50) after intravenous administration to rats was characterized. While a rapid elimination from the systemic circulation was observed for MS-50, much more prolonged circulating property was observed for LMS-50. In addition, this in vivo disposition property of LMS-50 was suggested to be ascribed to its lower affinity to the liver, which is the determining organ of the in vivo disposition of MS-50. The evaluation of surface hydrophobicity of MS-50 and LMS-50 in buffer solution revealed that the surface of MS-50 is more hydrophobic than that of LMS-50. However, LMS-50 was oppositely found to be more hydrophobic than that of MS-50 in rat serum. The profiles of serum proteins associated with MS-50 and LMS-50 were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the amounts of some adsorbed proteins are greatly different between MS-50 and LMS-50. From these findings, it was suggested that the substantial difference in the in vivo disposition between MS-50 and LMS-50 would not be attributed to the difference in their surface hydrophobicity in the blood, but the difference in the type of serum proteins associated with them.     

Pharmacokinetic disposition of isoxicam in hepatic cirrhosis

To determine the effect of liver dysfunction on the absorption and disposition of isoxicam, we administered a single oral dose of 200 mg to 8 patients with alcoholic cirrhosis, and followed the plasma concentration-time curve for 96 h. The calculated isoxicam disappearance t 1/2 of 30 +/- 19.1 h in these patients was not different from the mean value of 31 +/- 5.7 h obtained in 10 normal subjects studied earlier by our group; however, interindividual variation was larger. No differences in lag time (0.42 +/- 0.3 vs 0.49 +/- .2 h) or time to peak concentration (9.0 +/- 2.5 vs 10.0 +/- 2.9 h) were observed. However, the peak plasma isoxicam concentrations and the areas under the plasma concentration-time curves (AUC) were reduced 32% (p less than 0.01) and 41% (p less than 0.01), respectively. Plasma protein binding of isoxicam studied by equilibrium dialysis was 80 +/- 16% in the cirrhotic patients compared with 96 +/- 1.4% in the normal volunteers (p less than 0.02), again with larger interindividual variation noted in patients with cirrhosis. The binding did not correlate significantly with any laboratory tests of liver function or with the AUC for plasma isoxicam. As compared to the normal subjects, these cirrhotic patients had decreased plasma isoxicam binding, peak plasma isoxicam concentrations and AUC, without a significant change in the apparent disappearance half-life of total plasma isoxicam after a single oral dose.     

Studies on the fat emulsion Intralipid. I. Association of serum proteins to Intralipid triglyceride particles (ITP)

Intralipid triglyceride particles (ITP) were isolated by density gradient centrifugation. The isolated ITP fraction contained about 70% of the total triglycerides and 20% of the total phospholipids of the original Intralipid emulsion. Intralipid emulsion was mixed with equal parts of either albumin solution or VLDL-free serum and the ITP immediately isolated by centrifugation. The isolated ITP contained protein of which more than 90% disappeared upon recentrifugation in the case of albumin and only to 20% in the case of serum. It was demonstrated by polyacrylamide disc gel electrophoresis and double immunodiffusion with monovalent antisera against apolipoproteins that the proteins associated with ITP were mainly albumin; apolipoproteins CII and CIII in the two types of experiments respectively. The specific association of apolipoproteins CII and CIII to ITP offers an explanation to the chylomicron like metabolism of Intralipid in vivo.     

Origin and nature of the proteins of bile. II. A comparative analysis of serum, hepatic lymph and bile proteins in the dog

Abstract. In the dog, as shown before in the human, the major plasma proteins in hepatic bile (albumin, orosomucoid, transferrin and immunoglobulin IgG), with the exception of immunoglobulin IgA, were found to be derived entirely from the plasma. Biliary IgA was shown to have two sources, the plasma and local biosynthesis. These conclusions were based on comparisons on specific radioactivities of different proteins in plasma and bile, after intravenous injection of canine plasma whose proteins had been labelled biosynthetically with tritium. — The transfer of plasma proteins from blood to bile was found to proceed along two parallel pathways, one involving bulk transfer of unmodified plasma, and one based on molecular sieving favouring the transfer of plasma proteins of low molecular weight. — From a comparative study of hepatic lymph and plasma, it was concluded that the molecular sieve process was completed by the time the plasma proteins reached the interstitial fluid. The sieving membrane was therefore assigned anatomically to the vascular walls, which were found to have the properties of an isoporous membrane with a pore radius of 102 Å. No further molecular sieving was found to take place during the crossing of the second barrier between blood and bile, viz. the epithelial sheet separating interstitial fluid from the bile.     

Evaluation of route of input on the hepatic disposition of diazepam

Diazepam, a drug of high intrinsic clearance, was studied in the in situ rat liver dually perfused with Krebs-bicarbonate buffer containing human serum albumin (HSA; 0-1%) and unlabeled diazepam (1 mg/l) under constant hepatic arterial (3 ml/min) and portal venous (PV; 12 ml/min) flow rates. Events after a unit impulse (using [(14)C]diazepam) and at steady state (using unlabeled diazepam) were evaluated. In the absence of HSA the fractional effluent recovery (F) after hepatic arterial infusion (0.046 +/- 0.013) was about twice that after PV infusion (0.019 +/- 0.006). With HSA present, regardless of input route, F increased as unbound diazepam fraction in perfusate decreased (e.g., for PV, F = 0.58 +/- 0.05 and 0.69 +/- 0.02 for unbound diazepam fraction values of 0.18 +/- 0.01 and 0.037 +/- 0.01 at 0.25% and 1% HSA). The effluent [(14)C]diazepam profile was also dependent upon HSA. On decreasing HSA from 1 to 0.25% the early sharp peak (at 12-20 s) was replaced by a flatter unimodal profile with a later peak (at 60-80 s). Comparison of estimated effective permeability-surface area product to perfusate flow ratios (4.4 for 1% HSA and 21 for 0.25% HSA) indicated a shift from a perfusion rate-limited uptake with 0.25% HSA to one intermediate between permeability and perfusion at 1% HSA. Recognizing that orally absorbed drug enters the liver only via PV and i.v. drug via both vascular routes, this study emphasizes the difference in hepatic extraction of compounds depending on route of input, and the role that alteration in perfusate binding has on hepatic drug disposition.     

Glycated proteins in serum: effect of their relative proportions on their alkaline reducing activity in the fructosamine test

The fructosamine test is considered clinically useful for assessing short-term integrated control of blood glucose, but there are few published data to support this hypothesis. We fractionated glycated and nonglycated proteins by affinity chromatography on phenylboronate columns and, with specific immunochemical methods, determined in the eluted fractions the following proteins, selected according to their biological half-lives and relative concentrations in serum: albumin, IgA, IgG, IgM, apolipoprotein B, haptoglobin, transferrin, alpha 1-antitrypsin, and alpha 2-macroglobulin. We found the following correlations between fructosamine (mmol/L) and, respectively, glycated albumin, IgG, and (albumin + IgG) (each in grams per liter): r = 0.901, 0.702, 0.878. IgM had the highest percentage of glycated molecules (range 11.1-37.5%, mean 22.4%), haptoglobin and alpha 1-antitrypsin the least. This result was almost independent of the proteins' molecular masses and fractional catabolic rate. Albumin evidently contributes most to results of the fructosamine test, confirming conclusions obtained in different ways by others.     

Heat shock proteins and their role in heart injury

Heat shock protein (HSP) synthesis arises transiently as a tool to protect cellular homeostasis after exposure to heat and a wide spectrum of stressful and potentially deleterious stimuli. HSPs are "molecular chaperones" that recognize and form a complex with incorrectly folded or denatured proteins, which ultimately leads to correct folding, compartmentalization, or degradation. Accumulating evidence has implicated HSPs as mediators of myocardial protection, particularly in experimental models of ischemia and reperfusion injury. Impaired myocardial performance, which results from many factors, including hypoxia, is one of the main mechanisms responsible for heart failure in the critically ill patient. In this setting, different protective functions have been attributed to HSPs, which include repairing ion channels, restoring redox balance, interacting with nitric oxide-induced protection, inhibiting proinflammatory cytokines, and preventing apoptosis pathway activation. On this basis, novel therapeutic strategies by means of promising pharmacologic interventions and/or gene transfection techniques are being investigated for their potential to enhance HSP expression by myocardial cells, with the goal of improving the outcome of the critically ill patient.     

血清中游离乙型肝炎表面抗原的意义

在乙型肝炎患者的血清中存在大量的没有感染性的亚病毒颗粒,这些亚病毒颗粒是由乙型肝炎表面抗原组成的小球形或管型颗粒,其浓度可达到病毒颗粒的100000倍,但对于过量的亚病毒颗粒的产生及其生物学功能目前仍不完全清楚。本文就HBV亚病毒颗粒的生成及其在病毒致病性和临床抗病毒治疗方面的意义进行了阐述。     

Biological activities of Junin virus proteins. II. Complement-fixing polypeptides associated with the soluble antigen and purified virus particles

In the Junin virus-infected cell, there is a soluble antigen (SAg), which we have detected by complement fixation. PAGE of purified SAg revealed two polypeptides with molecular weights of 20,000 and 25,000. An antigenically identical component, which was demonstrated by complement fixation and immunodiffusion tests and which had the same electrophoretic profile as SAg, was found after proteolytic breakdown of purified virions. SAg is an internal component of the virion, since it is unable to stimulate neutralizing antibodies and it can be obtained from viral particles whose envelopes have been removed by Triton X-100 treatment. The antigenic determinant apparently resides in the major nonglycosylated polypeptide (nucleoprotein P64), since the proteolytic degradation product is obtained in quantity and it is nonglycosylated.     

Effects of posture on concentrations of serum proteins in healthy adults. Dependence on the molecular size of proteins

The effect of posture on changes in protein composition of serum and its dependence on molecular size has been investigated in forty individuals classified by age and sex. Blood specimens were drawn (A) at 0700 hours with persons still resting in bed after an overnight sleep; (B) at 0815 hours, in sitting position after 1 h of moderate exercise and again (C) at 0915 hours, lying in bed after 1 h of recumbency. Concentrations of alpha 1-antitrypsin, albumin and alpha 2-macroglobulin in serum were determined with high analytical precision. The mean fractional increases of concentrations from lying (A) to sitting (B) were 0.069 for alpha 1-antitrypsin and 0.063 for albumin, but lower, 0.040, for alpha 2-macroglobulin. Thus these increases, being related to molecular size, were not simply caused by posture induced changes of plasma volume. Considering the results of each individual we found a constant difference between the increase of alpha 1-antitrypsin and alpha 2-macroglobulin of about 0.030 for the whole range of increases (i.e. for alpha 1-antitrypsin 0-0.15). In contrast, the effects of recumbency were independent of molecular size. The mean, fractional decreases in concentration induced by changing of position from sitting (B) to lying (C) were approximately 0.065 for all three proteins.