Rat Plasma Proteins Research Articles

Selective inhibition of arachidonate 5-lipoxygenase by novel acetohydroxamic acids: biochemical assessment in vitro and ex vivo.

1. The chemically novel acetohydroxamic acids, BW A4C, BW A137C and BW A797C, are potent inhibitors of the synthesis of leukotriene B4 (LTB4) from arachidonic acid by human leucocyte homogenates: the concentrations required for 50% inhibition (IC50) were 0.1 microM, 0.8 microM and 0.5 microM respectively. Inhibition was less at higher concentrations of arachidonic acid. 2. These compounds also inhibited the synthesis of [14C]-5-HETE from [14C]-arachidonic acid and the calcium-dependent synthesis of LTB4 from 5-HPETE. This, therefore, suggests that they inhibit 5-lipoxygenase and LTA4 synthase. 3. Concentrations of acetohydroxamic acids required to inhibit metabolism of arachidonic acid by cyclo-oxygenase, 12-lipoxygenase and 15-lipoxygenase were 10 to 100 times higher than those required to inhibit 5-lipoxygenase. 4. The compounds were potent inhibitors of LTB4 synthesis induced by the ionophore, A23187, in human intact leucocytes. This inhibition was reversed by washing the cells. They were also potent, selective inhibitors of LTB4 synthesis induced by A23187 in whole rat blood: binding to rat plasma proteins did not greatly reduce the effectiveness of the compounds. 5. The effects of the acetohydroxamic acids, administered either intravenously or orally to rats, on the synthesis of LTB4, and thromboxane B2 (TXB2) in A23187-stimulated blood ex vivo was studied. The three compounds caused dose-dependent inhibition of the synthesis of LTB4 but not TXB2. Inhibition of LTB4 synthesis persisted for up to 6 h after a single oral dose of 50 mg kg-1. 6. The plasma concentrations of unchanged compound determined by h.p.l.c. correlated with the inhibition of LTB4 synthesis ex vivo.

Characterization of enkephalin-immunoreactive peptides generated by peptic digestion of rat plasma proteins.

Met-Enkephalin-immunoreactive peptides (MEIP) and Leu-enkephalin-immunoreactive peptides (LEIP), generated from plasma proteins after digestion with pepsin, were characterized with gel filtration, reverse phase HPLC, and isoelectric focusing. Pooled rat plasma was found to generate 170 pmol LEIP and 76 pmol MEIP/ml plasma. Two peaks of enkephalin (enk)-immunoreactive peptides (EIP) were observed after gel filtration of plasma digests; the early eluting peak (which contained 90% of the total LEIP and 75% of the total MEIP) eluted close to, but not in, the void volume of the column, whereas the elution volume of a later eluting peak (which contained 25% of the total MEIP and about 5% of the total LEIP) was close to that of authentic enk. Five peaks of EIP with pI values of 3.7, 5.8, 7.3, 8.0, and 9.4, were observed after isoelectric focusing of plasma digests; with the exception of the pI 3.7 peak, all of these immunoreactive species could be detected with greater efficiency using the Leu-enk RIA. Peptic digestion of rat serum albumin generated a MEIP with the same pI and HPLC retention time as the plasma pI 3.7 MEIP. No other EIP could be generated from rat serum albumin. No immunoreactive peptides were found which coeluted with synthetic Met-enk or its sulfoxide on HPLC; however, a portion of low mol wt LEIP was eluted with the retention time of authentic Leu-enk. With certain exceptions, these results support and extend the original findings of Singer and Carraway concerning the ability of pepsin to generate extremely high concentrations of EIPs from plasma protein(s).

The interaction of aflatoxin B 1 with vitamin K, phenylbutazone, and sulfamethoxine in rats

The interactions of aflatoxin B 1 (AFB 1) with vitamin K, phenylbutazone, and sulfamethoxine were investigated in albino rats. Vitamin K (5 mg/kg) was able to completely suppress the increase in whole blood clotting time caused by AFB 1 (25 μg/kg). Phenylbutazone (50 mg/kg) and sulfamethoxine (50 mg/kg) also significantly ( P < 0.05) lowered the increased clotting time caused by AFB 1. Equilibrium dialysis was performed on rat plasma (4 mg/ml protein content) to investigate the displacement of AFB 1 (3 μg) from its bound form by vitamin K (250 μg), phenylbutazone (2500 μg), and sulfamethozine (2500 μg). Phenylbutazone and sulfamethoxine significantly ( P < 0.05) displaced AFB 1 from rat plasma protein. Histopathological examinations performed on the liver, kidneys, and spleen of control and treated rats showed that none of the drugs used appeared to offer any significant organ protection against AFB 1 except in the spleen.

Drug-protein conjugates—XV: A study of the disposition of D-penicillamine in the rat and its relationship to immunogenicity

The disposition of [ 14C] d-penicillamine (PA) was investigated in vitro and in vivo with male Wistar rats. Irreversible binding of [ 14C]PA to isolated rat plasma proteins in vitro reached a maximum of 20.6% of total radioactivity at 6 hr. Irreversibly bound [ 14C]PA could be dissociated with dithiothreitol, demonstrating that conjugation was via disulphide linkage. Three hours after i.v. administration of [ 14C]PA (27 μmol/kg) to rats 100% of plasma radioactivity was irreversibly bound, representing approximately 3.5% of the dose. Further studies on the disposition of PA-plasma protein conjugates snowed that dissociation occurred readily in vivo: the plasma half-life of the conjugate was approximately 3 hr. Free [ 14C]PA was the major urinary metabolite after administration of both free and conjugated drug. These studies show that the disposition of PA is similar to that reported for the structurally related sulphydryl drug captopril (CP). Free PA (340 μmol/kg and 3.4 mmol/kg) administered i.p. and i.m. daily for 4 days at one monthly intervals, and also PA-KLH conjugate (100 μg/rat) administered by single i.p. injection at monthly intervals with and without Freund's complete adjuvant, failed to induce PA-specific IgG or IgM antibody responses detectable by ELISA. In contrast, CP (270μmol/kg), administered by the same protocol as free PA, induced a CP-specific IgG antibody response after the third series of monthly injections. These data suggest that the difference in immunogenicity between PA and CP arises from a difference in the intrinsic immunogenicity of the haptens, rather than from their disposition.

Binding of glycyrrhetinic acid to rat plasma, rat serum albumin, human serum, and human serum albumin.

The binding of glycyrrhetinic acid (GLA) to rat plasma, rat serum albumin (RSA), human serum, and human serum albumin (HSA) was examined by an ultrafiltration technique. Specific and nonspecific bindings were observed in all cases. The assosiation constant (K) for the specific binding was 1.34 × 105 M-1 in rat plasma and 1.56 × 105 M-1 in RSA. The K value for human serum (14.91 × 105 M-1) and that for HSA (15.09 × 105 M-1) were approximately 10-fold larger than those for rat plasma and RSA, suggesting a species difference in the specific binding affinity for GLA. The number of specific binding sites (n) was 2.49 and 1.18 for RSA and HSA, respectively. The linear binding coefficient (ψ) for the nonspecific binding was 7.11 × 103 M-1 in RSA and 6.57 × 103 M-1 in HSA.When the rat plasma and human serum protein concentrations are respectively assumed to be 3.5% and 4.2% (equal to the measured plasma and serum albumin concentrations), n was 1.83 in rat plasma and 1.31 in human serum, which corresponded fairly well to those for RSA and HSA. Further, for ψ in rat plasma and human serum, when rat plasma and human serum protein are the measured albumin concentrations, respectively, ψ was 10.97 × 103 M-1 for rat plasma and 14.83 × 103 M-1 for human serum, which corresponded well to those for RSA and HSA.It was concluded that the GLA-binding sites in rat plasma and human serum exist in albumin and GLA binds to specific and nonspecific binding sites at lower and higher concentrations than approximately 2 mM, respectively.

Selenite metabolism in rat and human blood

The binding of selenite, the form of selenium used in the treatment of Keshan disease, to plasma proteins and the role of erythrocytes in this process have been studied. The experiments were carried out by incubating 75Se as selenite with plasma and whole blood in vitro (human and rat) and in vivo (rat) and subsequent fractionation by Sephadex G-150 gel filtration. Human and rat plasma proteins were unable to incorporate selenium from selenite, as shown by the negligible amount present in proteins after incubation of plasma with selenite. The incorporation can be carried out after internalization of selenite by the erythrocytes in a fast, temperature dependent process. Hemoglobin, being the major binding protein for the newly reduced selenium in the erythrocyte, might have a role in the uptake of selenite by erythrocytes. The greater affinity of plasma proteins for the final selenium compound resulting from reduction could be the cause of the efflux. However, this is minimal in the absence of plasma, as is evident from the results obtained from blood reconstituted with saline solution instead of plasma. At least two proteins, one albumin like, probably albumin itself and the other of molecular weight close to or greater than 200,000 in the plasma, are involved in the binding and efflux processes.

Binding of 1-(2-ethoxyethyl)-2-(hexahydro-4-methyl-1,4-diazepin-1-yl)benzimidazole difumarate (KG-2413) to serum or plasma proteins in man, guinea pig and rat.

Human, guinea pig and rat sera or plasma protein binding of 1-(2-ethoxyethyl)-2-(hexahydro-4-methyl-1, 4-diazepin-1-yl)benzimidazole difumarate (KG-2413) were investigated by equilibrium dialysis. There were pronounced interspecies differences in binding affinity and binding capacity which were the highest in guinea pig and human species, respectively. In humans, the binding index (n1P1/Kd1) of isolated alpha 1-acid glycoprotein (alpha 1-AG) was seven times higher than that of isolated albumin, and the dissociation constant for human serum at the high affinity binding site was comparable to that for alpha 1-AG. These facts suggested that the free base of KG-2413 was mainly bound to alpha 1-AG in human serum. The contribution of albumin to the binding of KG-2413 in guinea pig and rat plasma may be relatively small. The interspecies differences in KG-2413 binding observed in this study may be due to species differences in the binding affinity and/or capacity of alpha 1-AG.

Plasma binding of 1-butanol, phenol, nitrobenzene and pentachlorophenol in the rainbow trout and rat: A comparative study

1. 1. The in vitro binding of 1-butanol, phenol, nitrobenzene, and pentachlorophenol in trout plasma and rat plasma was determined. 2. 2. Binding to rainbow trout plasma proteins agreed within 9% of that observed in rat plasma. 3. 3. Percentage bound to rainbow trout (2–99%) or rat (10–99%) plasma proteins increased as the log octanol/water partition coefficient of the chemicals increased within the Log P 1–3 range, and was suggestive of hydrophobic interactions in binding.

In vitro stability, plasma protein binding and blood cell partitioning of 14C-carboplatin.

Radiochemically pure 14C-labeled carboplatin, cis-diammine [1,1-cyclobutane (1-14C) dicarboxylato (2-)-0,0'] platinum (II), was added to fresh human, dog and rat plasma, at concentrations ranging from 1 to 100 micrograms 14C-carboplatin/ml. After 10 min incubation at ambient temperature, the plasma was ultrafiltered in Amicon Centrifree micropartition units to generate protein-free plasma ultrafiltrate (PU). Total radioactivity was determined by liquid scintillation counting. A mean (+/- SD) of 102% +/- 2.0%, 99.5% +/- 1.9%, and 99.0% +/- 1.0% of the 14C-carboplatin added to fresh human, dog, and rat plasma respectively was recovered in the PU. 14C-carboplatin was incubated at 37 degrees C with fresh plasma (60 micrograms/ml) and urine (200 micrograms/ml) from humans and dogs for 120 h, and samples were removed at appropriate times for analysis of carboplatin, 1,1-cyclobutane dicarboxylic acid and cyclobutane carboxylic acid. The latter were separated by HPLC on a C-18 column with a mobile phase of H2O/CH3CN/0.3 M tetrabutylammonium phosphate (880:50:20 v/v/v), and the column eluants at the retention time of each compound were collected and counted for total radioactivity. Carboplatin degraded in each of the matrices with a corresponding release of 1,1-cyclobutane dicarboxylic acid. 14C-carboplatin (50 micrograms/ml) was incubated at 37 degrees C with fresh human, dog and rat blood and the distribution of radioactivity into the cellular fraction was determined. Radioactivity did not distribute into the blood cells of humans or dogs, but after 5 h, 44% of the radioactivity in rat blood was associated with the cellular fraction. These results show that carboplatin, at physiological concentrations, does not bind instantaneously and reversibly to the plasma proteins of rat, dog or human, and that the molecule slowly degrades in plasma and urine in vitro with the release of 1,1-cyclobutane dicarboxylic acid. The remaining diammine platinum (II) portion of the molecule therefore accounts for the essentially irreversible protein binding of the platinum from carboplatin.

Binding of Estradiol-17-Fatty Acid Esters to Plasma Proteins*

The C-17 fatty acid esters of estradiol are a unique family of long-acting estrogens that circulate in blood. The unusual duration of the estrogenic action of these esters has been shown previously to correlate with their very slow rate of metabolism. However, in striking contrast to their slow rate of metabolism, the clearance of these esters from blood is relatively rapid, not very different from that of estradiol (E2). Studies on the effect of the size of the carboxylic acid moiety on the rates of both metabolism and clearance have suggested that an active process might be involved in the cellular uptake of these circulating esters, and this, in turn, raised the question of how E2-fatty acid esters are transported in blood. The binding of representative E2-17-fatty acid esters to both human and rat plasma proteins known to bind either E2 or fatty acids was investigated. As expected, both E2 and 5 alpha-dihydrotestosterone bound to human sex hormone-binding globulin, whereas none of the E2 esters bound to this human plasma protein. Similarly, E2 bound to rat alpha-fetoprotein (AFP) and unsaturated fatty acids bound to both human and rat AFP, but none of the E2 esters bound to AFP of either species. These steroid esters bind to lipoproteins. Over 85% of a representative ester, E2-17-stearate, partitioned in the lipoprotein fractions of both human and rat serum, while the synthetic short chain ester, E2-17 beta-acetate, like E2 itself, partitioned predominantly in the nonlipoprotein fraction of blood. These results demonstrate the unusual binding of a family of steroid hormones to plasma lipoproteins and open the possibility that they are transported into target cells through the mediation of lipoprotein receptors.

High-performance liquid chromatographic analysis, plasma protein binding and red blood cell partitioning of phenprobamate.

A new high-performance liquid chromatographic procedure for the analysis of phenprobamate, a skeletal muscle relaxant in biologic fluids was developed. The method used a C18 reverse phase column, a mobile phase of methanol/acetonitrile/water (33:15:52), and UV detection at 215 nm. The assay procedure was applied to the determination of phenprobamate binding to rat and human plasma proteins using the equilibrium dialysis method. In addition, the red blood cell/plasma partitioning was determined in the whole blood of rats and humans. Phenprobamate exhibited a moderate binding to plasma proteins of rat (74.3 +/- 2.2 per cent) and human (80.5 +/- 1.1 per cent). The protein binding was concentration-independent in the range of 10 to 80 micrograms ml-1. Phenprobamate binding to plasma proteins was also determined in the presence of 10 micrograms ml-1 acetaminophen. The protein binding of phenprobamate was not significantly altered by acetaminophen (74.4 +/- 0.6 per cent for rat plasma; 75.7 +/- 1.6 per cent for human plasma). The distribution ratios of phenprobamate between the red blood cells and plasma were greater than unity, 1.86 and 1.59 in rat and human, respectively, indicating a preferential partitioning of the drug in the red blood cells.

Changes in plasma proteins in rats treated for short periods with hepatotoxins or with agents which induce cytochrome P450 isoenzymes.

We have compared the alterations in plasma proteins following treatment of rats with the centrilobular hepatotoxin carbon tetrachloride, the periportal hepatotoxin allyl alcohol and two inducers of hepatic microsomal drug metabolising enzymes, phenobarbitone and a mixture of polychlorinated biphenyls (Aroclor 1254). The results are compared with changes observed in animals treated with agents which caused proliferation of rat liver peroxisomes and described in an earlier publication. Our results indicate that: There is a marked induction of a minor glycoprotein which migrates as a prealbumin following treatment with both inducers of microsomal mixed function oxidases and inducers of peroxisome proliferation. There is a marked increase in a minor alpha 1-glycoprotein when there is chemically-induced mitosis in the liver but not in mitosis following liver damage. Two major alpha 1-glycoprotein are depressed in all forms of liver damage. There are indications of specific protein responses to allyl alcohol, to inducers of microsomal mixed function oxidases and to inducers of peroxisome proliferation.

Drug-protein conjugates—XII a study of the disposition, irreversible binding and immunogenicity of penicillin in the rat

The disposition, irreversible binding and immunogenicity of benzylpenicillin (BP†) were studied in male Wistar rats. [ 3H]BP, administered i.v. to anaesthetized rats at two doses (27 μmol/kg, 2.7 mmol/kg), showed dose-dependent kinetics: plasma and tissue concentrations of total BP were disproportionately increased at the higher dose. BP was rapidly cleared from the plasma at both doses (<0.05% of administered dose/ml plasma after 3 hr). In spite of the disproportionately elevated levels of total BP after the higher dose, covalent binding to plasma proteins was quantitatively similar as a percentage of the dose at both doses. Three hours after i.v. injection of 27 μmol/kg and 2.7 mmol/kg of the drug, 5.6% ± 1.7% and 3.3% ± 1.1% respectively of circulating BP was covalently bound, representing <0.004% of the administered dose bound per ml of plasma in each case. Covalent binding of BP to rat plasma proteins in vitro was of a similar magnitude to that observed in vivo: 1.6% ± 0.4% of BP was bound to 25% rat plasma after 3 hr incubation at 37°. In a separate series of experiments the immunogenicity of BP was studied by chronic administration of the drug to rats. Following daily i.v. or i.m. administration of BP (27 μmol/kg, 270 μmol/kg, 2.7 mmol/kg) for 4 consecutive days at 4-week intervals (three series of injections) neither IgG nor IgM anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Intravenous administration of the high dose of BP was discontinued after the first series of injections due to local necrosis. In contrast to free BP, BPO-keyhole limpet haemocyanin (BPO-KLH, 42 nmol BP bound/mg KLH) administered by single i.v. injection at 4-week intervals at two doses (20 and 200 μg conjugate/kg, corresponding to 0.84 and 8.4 nmol BPO/kg) readily induced IgG and IgM anti-BPO antibody responses (median IgG titres were 872 and 5470 one week after the third injection of the low and high dose of conjugate respectively; corresponding IgM titres were 4513 and 22,866). The specificity of the IgG and IgM antibodies for the BPO determinant was confirmed by ELISA inhibition with BPO-aminocaproate. These experiments show that BP binds irreversibly, but to a limited extent, to plasma proteins in vivo, and that such a degree of conjugation appears to be insufficient to elicit a detectable anti-BPO antibody response.

Studies on the metabolic disposition of ethyl eicosapentaenoate(EPA-E) in rats and dogs.

Plasma level, tissue distribution and excretion of ethyl eicosapentaenoate(EPA-E) were studied after oral administration of 14C-uniformly labelled EPA-E to rats and dogs and the following results were obtained. 1. The maximum concentration of radioactivity in plasma was obtained at 6-9 hr and at 24hr after dosing in rats and dogs, respectively. The elimination of radioactivity from plasma was biphasic. 2. The radioactivity administered to rats was mainly excreted in expired air. 3. The cumulative biliary excretion of radioactivity was 2.9 % of the dose within 24 hr. 4. The concentration of radioactivity in each tissue reached maximum at 9-24hr after dosing except for brain, fat, skin, muscle and aorta in which the concentration reached maximum at 1 week and subsequently decreased gradually. The elimination rate of radioactivity from each tissue was almost as same as that from plasma or a little slower. 5. Foeto-placental transfer of radioactivity in the pregnant rats at 12 and 19 days of gestation was observed but its extent was low. 6. The milk level of radioactivty in the lactating rats reached maximum at 24 hr and was 6-14 times higher than that in the maternal blood after 24hr. 7. Radioactive substances in plasma were mainly bound to plasma protein in rats and dogs. 8. During daily repeated administration to rats, the concentration of radioactivity in plasma at 24hr after every dosing reached plateau level within the 12 days but that in each tissue increased with increase in the number of doses. No remarkable difference in elimination half-lives of radioactivity in each tissue between the single and repeated dosing was observed.

Potential mechanisms for redistribution of polychlorinated biphenyls during pregnancy and lactation.

Female mice treated with 14C-2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) two weeks prior to mating eliminated virtually their entire body burden of the compound through milk during one lactation cycle. 6-CB was shown to distribute among rat and human plasma lipoproteins and protein in vitro. It was readily transferred among plasma constituents and its distribution was related to the triacylglycerol:protein ratio in plasma. At one hour following its intravenous administration to virgin rats, 6-CB was primarily distributed to LDL. With the hypertriglyceridemia of late pregnancy, more than 70% of circulating 6-CB was associated with VLDL. VLDL is a major substrate for mammary gland lipoprotein lipase which is elevated during lactation. When 6-CB was complexed with human VLDL and injected i.v. into late pregnant mice, mammary gland concentrations of 6-CB exceeded those of adipose tissue at all sacrifice times between 5 min and 6 h. No differences between adipose tissue and mammary gland concentrations of 6-CB were observed with Emulphor:ethanol:saline as vehicle until 6 h. Isolated hepatocytes were capable of secreting protein and triacylglycerol in the form of VLDL into serum-free media. Eighty percent of 6-CB released from hepatocytes was in association with VLDL, with the remainder in association with protein. Adipocytes isolated from epididymal fat pads of male rats which were pretreated with 6-CB released progressively less radioactivity to incubation media with time after treatment even though PCB content of these cells increased. 6-CB may not be evenly distributed among adipocyte lipids.

Lipophilicity and disposition of 1-aryl-piperazines in the rat.

The disposition of eight 1-aryl-piperazines was investigated in rats after i.v. administration. The concentration of 1-aryl-piperazine in body fluids and tissues was determined by h.p.l.c. or electron-capture g.l.c. Correlations of kinetics and physiological parameters with the lipophilicity of each 1-aryl-piperazine, determined by h.p.l.c. retention on a reverse-phase C18 column at neutral pH, were investigated. Binding to rat plasma proteins varied within the series, increasing with lipophilicity. For the majority of the derivatives the blood-to-plasma ratio was close to unity, implying an almost equal distribution between erythrocytes and plasma. The most lipophilic 1-aryl-piperazine of the series partitioned more into erythrocytes. The eight compounds differed widely in Vss and total Cl values and as a general trend both values increased with lipophilicity. The percentage of the dose excreted unchanged in the urine decreased progressively with increasing lipophilicity. 1-Aryl-piperazines were distributed extensively in all the tissues examined, concentrating particularly in the eliminating organs and lung. They easily entered the rat brain, Cmax values generally being reached within five minutes of parenteral injection. 1-Aryl-piperazine brain uptake increased with lipophilicity.

Measurement of Glycosylated Haemoglobins and Glycosylated Plasma Proteins in Animal Models with Diabetes or Inappropriate Hypoglycaemia

Stable glycosylated haemoglobins and glycosylated plasma proteins were determined by affinity chromatography using Glycogel B in animal models with diabetes or inappropriate hypoglycaemia. Adult Aston ob/ob mice and C57BL/KsJ db/db mice exhibited 1.5-1.9 fold increases of body weight, 2.5-3.4 fold elevations of plasma glucose and 20.9-29.3 fold elevations of plasma insulin. Glycosylated haemoglobins and glycosylated plasma proteins were raised 7.2-8.2 fold and 6.6-6.7 fold respectively. In adult NEDH rats, administration of streptozotocin or implantation of transplantable insulinoma fragments produced reciprocal changes in insulin and glucose concentrations either resulting in the onset of insulin deficiency (5.9 fold decrease) and hyperglycaemia (3.2 fold increase) by 2 days, or hyperinsulinaemia (2.1 fold increase) and hypoglycaemia (1.4 fold decrease) by 6 and 8 days, respectively. Glycosylated plasma proteins were increased (1.2 fold) rapidly after streptozotocin treatment followed by glycosylated haemoglobins (1.6 fold increase) after 8 days. In contrast, the decreases in glycosylated plasma proteins and glycosylated haemoglobins (4.4 fold and 1.4 fold, respectively) in insulinoma-bearing rats preceded the demonstration of hypoglycaemia by 5 and 2 days, respectively. Glycosylated plasma proteins in insulinoma-bearing rats returned to pretransplantation values at 10-16 days. Good correlations were observed in mice and rats between glucose and both glycosylated haemoglobins (r = 0.92 and r = 0.90, respectively) and glycosylated plasma proteins (r = 0.85 and r = 0.93), and between the glycosylated blood proteins themselves (r = 0.95 and r = 0.91). The results show that the measurement of glycosylated blood proteins by affinity chromatography using Glycogel B provides a sensitive and reliable indicator of the recent glycaemic environment.

The effect of the nucleoside transport inhibitor dipyridamole on the incorporation of [3H]thymidine in the rat

Dipyridamole is a non-specific inhibitor of nucleoside transport into mammalian cells. It is currently undergoing clinical evaluation in combination with various antimetabolites in an attempt to enhance the activity of these anticancer drugs by blocking the salvage of extracellular nucleosides, an important determinant of their cytotoxicity. In the present study, the effect of i.v. infusions of dipyridamole on [3H]thymidine incorporation into DNA has been examined in the anaesthetized rat. The tissues studies were bone marrow, gastrointestinal tract epithelium and the ascitic form of the Walker carcinosarcoma. Dipyridamole at 10 mg/kg, given over 3 hr, led to plasma levels of < 5 μM and did not reduce [3H]thymidine incorporation into any of the tissues studied. At 40 mg/kg dipyridamole (plasma levels 10–15 μM) [3H]thymidine incorporation into the DNA of bone marrow and gastrointestinal tract epithelium was reduced to 20–30% of control values. Increasing the dose to 100 mg/kg did not lead to a further suppression of incorporation. Measurement of [3H]thymidine plasma pharmacokinetics and the intracellular distribution of tritium suggested that the inhibition of [3H]thymidine incorporation was due to reduced cellular uptake. In contrast to the effects on normal tissues, even at a lethal dose (200 mg/kg) dipyridamole did not significantly inhibit [3H]thymidine incorporation into Walker tumour cells. The levels of dipyridamole found in the ascitic fluid, at 100 mg/kg approximately half those in plasma, argue against a pharmacokinetic basis for this difference. Dipyridamole was found to bind extensively (97%) to rat plasma proteins, which may explain the discrepancy between the concentrations of dipyridamole required to inhibit nucleoside incorporation in vitro, in serum-free media, and those needed in vivo. From a comparison of the plasma levels of dipyridamole which cause an inhibition of [3H]thymidine incorporation in the rat with those which can be achieved safely in patients, it is concluded that dipyridamole is unlikely to markedly reduce nucleoside salvage in man.

Contribution of plasma proteins to the vitreous of the rat.

The extent of plasma proteins in the vitreous humor of the rat was determined. Disc gel electrophoresis of the soluble proteins of the rat vitreous humor showed many protein bands, but most of the bands did not coincide with those of rat plasma. Using antibodies to rat plasma proteins, immunological analyses of vitreous and plasma, including fractions separated by high-pressure gel permeation chromatography, showed that several vitreous proteins react with plasma protein antibodies. However, only albumin and transferrin were found to be completely identical in both fluids. The concentration of albumin in vitreous was about 625 micrograms/ml and that of transferrin was 61.6 +/- 19.1 (S.D.) microgram/ml. The total protein concentration in vitreous was 9.5 +/- 0.5 (S.D.) mg/ml. The ratio of transferrin to albumin in the vitreous was approximate to that of plasma, but the sum of the two proteins accounted for less than 10% of the total protein. These data indicate that in the rat some plasma proteins, particularly albumin and transferrin, are found in the vitreous, but most of the soluble proteins appear to originate from sources other than plasma, such as the vitreous itself or in surrounding tissues including the ciliary body and the retina.

Messenger ribonucleic acid (mRNA) from developing rat cerebellum directs in vitro synthesis of plasma proteins

Poly(A)-containing messenger RNA from different ages of postnatal rat cerebellum was translated in the reticulocyte lysate system, using [ 35S]methionine to label newly synthesized polypeptides. Translation products were identified using crossed immunoelectrophoresis and antisera against whole rat plasma and/or specific plasma proteins, followed by autoradiography. It was found that postnatal cerebellar mRNA directs the synthesis of low density lipoprotein (LDL), fibrinogen, transferrin, α 1-macroglobulin, α 2-macroglobulin and probably also α-fetoprotein, albumin and α 1-lipoprotein. mRNAs for several other yet undefined plasma proteins were also detected. In general, the synthesis of plasma proteins appeared to decline with cerebellar maturation. X-irradiation or hypothyroidism resulted in selective changes in the levels of mRNA-directed plasma proteins. In the X-irradiated cerebellum these included an increase in the level of mRNA for α-fetoprotein and a decrease in the level of mRNA for fibrinogen. In contrast, hypothyroidism decreased cerebellar plasma protein synthesis in general. These observations indicate that plasma proteins are actively produced within the developing rat cerebellum, and that the rate of their sysnthesis depends on the developmental stage and is influenced by the types of cells present.