Horse Plasma Research Articles

Acid-stable protease inhibiting polypeptides formed from denatured horse plasma by proteolysis

1. 1. Trypsin digestion of perchloric acid precipitated horse plasma yielded polypeptides with inhibitory properties for trypsin, chymotrypsin and, to a small extent, kallikrein. 2. 2. The M r of the inhibitory polypeptides were 73,000 and 24,000. 3. 3. The number, enzyme specificity and M r of the inhibitory polypeptides differed from the values known for the human being. 4. 4. The inhibitory polypeptides were purified by affinity chromatography on Sepharose—trypsin and by gel filtration through Sephadex G-75. 5. 5. Protease inhibitory polypeptides were generated in the same manner by chymotrypsin, elastase, proteinase K, pronase, collagenase, papain and subtilisin. 6. 6. The number and electrophoretic migration of the inhibitory polypeptides obtained with the different enzymes were variable. 7. 7. The enzyme specificity was constant since all polypeptides inhibited only trypsin, chymotrypsin and kallikrein to a small extent. 8. 8. None of the inhibitory polypeptides were immunologically related to native plasma proteins or plasma protease inhibitors.

Analysis of detomidine in horse blood, plasma and urine samples utilizing a sensitive gas chromatography—mass spectrometry method

Chemical ionization- an electron impact ionization-selective ion monitoring provided a simple and sensitive method for measuring detomidine (Domosedan), a potent sedative-analgesic drug for horses and cattle. Chemical ionization was at least 10 times more sensitive than electron impact ionization. By using propranolol as an internal standard, we found that the recovery of detomidine from the extraction procedure used in this study was greater than 75% for plasma, whole blood, or urine samples. Approximately 68% of detomidine was bound to plasma protein and 53% was bound to red blood cells.

Phenylbutazone in the horse: a review.

Phenylbutazone is an acidic, lipophilic, non-steroidal anti-inflammatory drug (NSAID). It is extensively metabolized in the horse. The metabolites so far identified, oxyphenbutazone, gamma-hydroxyoxyphenbutazone, account for some 25-30% of administered dose over 24 h. The plasma half-life of phenylbutazone and termination of its pharmacological action are determined primarily by its rate of hepatic metabolism. Phenylbutazone acts by inhibiting the cyclooxygenase enzyme system, which is responsible for synthesis of prostanoids such as PGE2. It appears to act on prostaglandin-H synthase and prostacyclin synthase, after conversion by prostaglandin-H synthase to reactive intermediates. It markedly reduces prostanoid-dependent swelling, edema, erythema, and hypersensitivity to pain in inflamed tissues. Its principal use in the horse is for treatment of soft tissue inflammation. Phenylbutazone is highly bound (greater than 98%) to plasma protein. After i.v. injection, blood levels decline with an elimination half-life of 3-10 h. The plasma kinetics of phenylbutazone may be dose dependent, with the plasma half-life increasing as the drug dosage level increases. Plasma residues of the drug at 24 h after a single i.v. dose of 2 g/450 kg average about 0.9 microgram/ml, but considerable variation occurs. If dosing is repeated, the plasma residue accumulates to give mean residual blood levels of approximately 4.5 microgram/ml on Day 5 after 4 days of dosing. Approximately similar blood levels are found after a combination of oral and i.v. dosing. Experiments on large numbers of horses in training have been undertaken to ascertain the population distributions of residual blood levels after such dosing schedules. Absorption of phenylbutazone from the gastrointestinal tract is influenced by the dose administered and the relationship of dosing to feeding. Access to hay can delay the time of peak plasma concentration to 18 h or longer. Under optimal conditions, the bioavailability of oral phenylbutazone is probably in the region of 70%. Paste preparations may be more slowly absorbed than other preparations and yield higher residual plasma levels at 24 h after dosing, but further controlled studies are required. Phenylbutazone is easily detected in the plasma and urine of horses but concentrations in saliva are low. It is quantitated for forensic purposes by HPLC. The variability of this method between laboratories is about +/- 25%. Increasing urinary pH increases the urinary concentration of phenylbutazone and its metabolites up to 200-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of mefenamic acid in horse plasma by high performance liquid chromatography.

A high performance liquid chromatographic procedure was developed for the quantitative analysis of mefenamic acid in horse plasma. To a 0.25 ml plasma sample, 25 μg of internal standard was added, and was extracted with methylene chloride under acidic conditions. Portions of the organic layer were evaporated to dryness under nitrogen gas. 'The residues were reconstituted in methanol and injected onto a column packed with Cosmosil 5C18. The elution was performed with a mixed solvent of methanol and 2% acetic acid (70 : 30, v/v) at 40°C. The flow rate was 2 ml/min, and the effluent was monitored at 352 nm. Mefenamic acid and the internal standard eluted at 8.8 and 11.2 min, respectively. The peak area ratio (mefenamic acid/internal standard) versus plasma concentration was linear in the range of 0.150μg/ml of mefenamic acid in plasma, and the detection limit of mefenamic acid was 0.05μg/ml. This method was successfully applied to the determination of mefenamic acid in the horse plasma after drug administration.

Determination of phenylbutazone and its metabolites in horse plasma and urine by high performance liquid chromatography.

ウマ血しょう及び尿中のフェニルブタゾン及び代謝物のオキシフェンブタゾン,γ-ヒドロキシフェニルブタゾンの高速液体クロマトグラフィーによる定量法を検討した.抽出溶媒はジクロルメタン又はベンゼンを使用した.カラムはCosmosil 5C18を用い,移動相は血しょうの場合,アセトニトリル-水-酢酸(55:42:3,v/v),尿では(49:48:3,v/v)を使用し,流速1.0ml/min,検出は254nmで行った.内標準物質はメテノロンを使用した.この移動相中ではγ-ヒドロキシフェニルブタゾンからカプロラクトン体への異性化が起こるが,これは試料を少量のトリエチルアミンを含む溶液に溶解することにより防止できた.検量線は3者とも血しょうで40μg/ml,又尿では160μg/mlまで直線性を示した.日内及び日差変動は血しょう,尿とも各々2%程度と良好であった.この方法をフェニルブタゾン投与ウマ血しょう及び尿中の各代謝物の濃度測定に応用した.

Evidence for the presence of two kininogens in porcine plasma and the isolation of high-molecular-weight kininogen.

When the pseudoglobulin fraction of porcine plasma was gel-filtered through a column of Sephadex G-150, two fractions, from which kinin was released on incubation with trypsin [EC3.4.21.4], were observed. This indicates the presence of two kininogens with different molecular weights in the plasma. The larger-molecular-weight kininogen fraction had the ability to correct the abnormal clotting time of human high-molecular-weight (HMW) kininogen-deficient plasma, Fitzgerald plasma. The smaller-molecular-weight kininogen fraction did not show this ability. Thus, the fraction showing the ability to correct the abnormal clotting time of Fitzgerald plasma was considered to contain a kininogen corresponding to that purified as an HMW kininogen from bovine, human, horse and rat plasma. The other fraction may contain porcine low-molecular-weight (LMW) kininogen.The porcine HMW kininogen was isolated by a modification of the purification method for horse HMW kininogen. The ability of the isolated HMW kininogen obtained to correct the abnormal clotting time of Fitzgerald plasma was in proportion to the amount of the preparation used. When this HMW kininogen preparation was incubated with purified porcine plasma kallikrein [EC 3.4.21.8], the release of bradykinin was observed.

Disposition of sulfadimidine and its N4-acetyl and hydroxy metabolites in horse plasma.

The plasma disposition of sulfadimidine (SDM) and its metabolites N4-acetylsulfadimidine (N4-SDM), 6-hydroxymethyl-4-methyl-pyrimidine (SCH2OH) and 5-hydroxy-4,6-dimethyl-pyrimidine (SOH), was studied in three horses following intravenous administration of SDM at dose levels of 20 and 200 mg/kg in cross-over trials. The percentages of N4-SDM (0.58-0.90%), SOH (0.83-6.75%) and SCH2OH (0.38-0.71%) in plasma, expressed as a percentage of the total sulfonamide concentration, were small and their plasma concentrations were parallel with SDM from 4 h following administration. At high doses (200 mg/kg), the elimination half-life was slightly longer than at low doses (6.0, 10.5, 11.0 vs 5.0, 9.5, 9.5, respectively). The plasma protein binding was related to the dose; it was for the 20 and 200 mg/kg doses, respectively:SDM:61.5-73.3% and 50.5-52.1%; SOH: 47.1-71.0% and 36.7-39.5%, and for N4-SDM: 45.9-63.2% and 38.3-53.7%. The protein binding for SCH2OH, measured in samples obtained at the high dose level, ranged from 13.8 to 20.0%.

Pre- α2-elastase inhibitor of the horse; a hybrid molecule between α1-proteinase inhibitor and α2- β1-glycoprotein

Pre- α 2-elastase inhibitor of horse plasma has recently been isolated in our laboratory. In this article we demonstrate that the inhibitor is a composite structure built of α 1-proteinase inhibitor and α 1- β 1-glycoprotein. The compound inhibitor is biologically active, although it has previously been shown that its enzyme specificity is different from that of free α 1-proteinase inhibitor. Our observations are based on immunochemical cross-reactions between pre- α 1-elastase inhibitor and antibodies to α 2- β 1-glycoprotein as well as antibodies to α 1-proteinase inhibitor. Pre- α 2-elastase inhibitor could be dissociated into its parent components by dithiothreitol. After dissociation, the α 1-proteinase inhibitor portion retained its biological activity.

Detection of staphylo-coagulase using plasmas from various animals

The effect of sources of Staphylococcus aureus and plasmas, concentration of plasma, temperature and duration of incubation on coagulase-test results was evaluated. Using S. aureus strains of food origin, the value of plasmas in coagulase tests was, in order of superiority, human and rabbit>pig>donkey>chicken>cattle>duck>goat>dog. However, with staphylococcal isolates of animal origin the order was cattle>pig>human> duck>goat>dog>rabbit>chicken>donkey. Regardless of the source of staphylococci, horse plasma was found unsuitable in coagulase tests as it clotted spontaneously. The temperature (25 and 37°C), and duration of incubation and type of anticoagulant had no significant ( P>0.05, X 2) effect on coagulase-test results. It is concluded that in testing staphylococcal isolates from various sources for coagulase production, it is imperative to use plasmas from several animal species whenever practicable as staphylococcal biotypes display variable ability to coagulate different plasmas.

An investigation, in vitro, of the actions of three Western Australian snakes on the blood coagulation of the dog, cat, horse and wallaby.

Venoms of the tiger snake and brown snake were procoagulant, in vitro, when tested with cat, dog, horse and wallaby plasma. In the absence of calcium and phospholipid the coagulant activity of tiger snake venom was minimal. In contrast, brown snake venom alone had marked procoagulant activity. This activity, however, was enhanced by the presence of calcium and phospholipid. Death adder venom exerted an anticoagulant effect. Apparent species' differences in susceptibility to the coagulant venoms were noted. However, the probable explanation of these differences was attributed to variation in the control values of the special studies rather than to a difference in the postulated actions of the venoms on prothrombin. A possible role for clotting studies in suspected snake bite in veterinary practice is suggested.

Multiple effects of isoproterenol on horse plasma cholinesterase

1. 1. Isoproterenol inhibits the hydrolysis of butyrylthiocholine by horse plasma cholinesterase, while it stimulates the hydrolysis of p-nitrophenyl butyrate. 2. 2. The inhibition pattern obtained for butyrylthiocholine is consistent with a dimeric model for the enzyme showing negative cooperativity. 3. 3. The kinetics of inhibition point to a dissociative effect of isoproterenol, superimposed on its competitive inhibitory action. The hydrolysis of p-nitrophenyl butyrate is not sensitive to changes in the subunit composition of the enzyme.

Substrate-dependent kinetic behavior of horse plasma cholinesterase: Evidence for kinetically distinct populations of active sites

The inhibition of horse plasma cholinesterase by propranolol showed characteristics which depended upon the identity of the substrate used. With butyrylthiocholine as substrate, the inhibition showed a first-order dependence on inhibitor concentration, and was characterized by a K i of 8 μ m (pH 7.4, 20 °C). With p-nitrophenylbutyrate as substrate, a biphasic v −1 versus [I] relationship was obtained. The biphasic curve could be resolved into two components, with apparent K i 's of 9 μ m and 1.3 m m. Use of butyrylthiocholine as alternative substrate resulted in partial inhibition of p-nitrophenylbutyrate hydrolysis. Inhibition of butyrylthiocholine hydrolysis by p-nitrophenylbutyrate could be accounted for by pure competitive inhibition at two sites. The results were interpreted in terms of a four-site, low-symmetry model, in which two active sites could process both substrates, and the remaining sites could process only p-nitrophenylbutyrate.

Laboratory and clinical evaluation of modified New York City medium (Henderson formulation) for the isolation of Neisseria gonorrhoeae

The growth-promoting properties of several ingredients of New York City (NYC) medium were investigated by using 100 recently isolated clinical strains of Neisseria gonorrhoeae. Difco GC medium base promoted the growth of large colonies of gonococci significantly better than other commercial GC medium bases. A 1% agar concentration resulted in the growth of larger colonies than 2% agar, without affecting the stability of the gel. Neither replacement of horse plasma with horse serum nor reducing the concentration of added serum from 12 to 3% affected the growth of even the most fastidious strains tested. From these observations, a modified NYC medium (Henderson) has been formulated which is easy to prepare and less expensive than NYC or Thayer-Martin medium. In a direct clinical comparison with 1,275 specimens, an isolation rate of 6.5% was achieved with both NYC medium and the Henderson formulation.

Effects of tryptamine antagonists on the anaphylactic contractions of the bovine pulmonary smooth muscles.

Calves were sensitized with horse plasma (H.P.), 0.2 ml/kg, i.v., and H.P. (0.2 ml/kg) in Freund's complete adjuvant, s.c. The latter injection was repeated 1 week later and the animals were killed 10 days after the second injection. Spirally cut strips of pulmonary artery and vein and the trachealis muscle from the sensitized calves contracted to 5-hydroxytryptamine (5-HT) and specific antigen (horse plasma). Antigen-induced contractions of the pulmonary smooth muscles were significantly blocked (P less than 0.05) by the 5-HT antagonists, methysergide and ketanserin. The trachea, however, appeared less sensitive to the antagonists than the pulmonary vessels. The results suggest that 5-HT participates in the pulmonary anaphylactic reactions of cattle and that ketanserin may be useful in suppressing bovine pulmonary hypersensitivities.

Determination of 4-aminopyridine in horse plasma using gas—liquid chromatography

Determination of 4-aminopyridine in horse plasma using gas—liquid chromatography

Qualitative detection of corticosteroids in equine biological fluids and the comparison of relative dexamethasone metabolite/dexamethasone concentration in equine urine by micro-liquid chromatography—mass spectrometry

Several important corticosteroids were qualitatively determined in the plasma and urine of horses by micro-liquid chromatography mass spectrometry (micro-LC—MS). The sensitivity and specificity of micro-LC—MS are demonstrated as is the ability of micro-LC— MS to deal with endogenous interferences. In turn, the relative amount of dexamethasone and its major unconjugated metabolite were determined in equine urine by micro-LC—MS; the conclusions drawn are reported.

Pharmacological properties of T-kinin (isoleucyl-seryl-bradykinin) from rat plasma

It is well known that trypsin liberates bradykinin from plasma. Recently we reported that, in addition to bradykinin, trypsin liberated from rat plasma isoleucyl-seryl-bradykinin which we termed “T-kinin” (1–3). T-kinin is a unique natural homolog of bradykinin because it does not contain the lysyl-bradykinin sequence which the known homologs from bovine (4), human (5), and horse (6) plasmas contain. In this communication we describe for the first time the pharmacological actions of T-kinin on the isolated guinea pig ileum, estrus rat uterus, and on the blood pressure of the rat.

Interspecies activation of lecithin-cholesterol acyltransferase by apolipoprotein a-i isolated from the plasma of humans, horses, sheep, goats and rabbits

The abilities of apolipoprotein A-I species isolated from humans, horses, sheep, goats and rabbits to activate purified human lecithin-cholesterol acyltransferase and the enzyme from homologous plasmas and plasma of other mammalian species were compared. Each purified apolipoprotein A-I species was individually incorporated into phosphatidylcholine cholesterol vesicles by the cholate dialysis method to form proteoliposome common substrates (apolipoprotein A-I/phosphatidylcholine/cholesterol molar ratio of 1:250:12.5) for the enzyme activity assay. All apolipoprotein A-I species tested had the ability, but with different effectiveness, to activate plasma lecithin-cholesterol acyltransferase from their own and other animal species and from purified human lecithin-cholesterol acyltransferase. Horse apolipoprotein A-I was nearly as effective as human apolipoprotein A-I, whereas apolipoproteins A-I from goats, sheep or rabbits were, in descending order, slightly less effective than either horse or human apolipoprotein A-I in activating human plasma lecithin-cholesterol acyltransferase. A similar trend in ability to activate purified human lecithin-cholesterol acyltransferase by these apolipoprotein A-I species was seen, but apolipoprotein A-I from sheep, goats and rabbits was only about 55–65% as effective as human apolipoprotein A-I. In general, apolipoprotein A-I from the same animal species as the lecithin-cholesterol acyltransferase enzyme was a better activator of the enzyme than was apolipoprotein A-I from another animal species. Comparison of lecithin-cholesterol acyltransferase activities of each plasma paired with its own apolipoprotein A-I, which reflects the level of the enzyme in the plasma, revealed descending levels of the enzyme's activity (nmol/h per ml) as follows: humans (98.9 ± 10.3), horses (90.7 ± 10.6), sheep (69.6 ± 9.7), goats (64.9 ± 9.2) and rabbits (52.0 ± 6.2). Studies with DEAE-Sepharose columns, gel electrophoresis and proteoliposome substrates demonstrated that apolipoprotein A-I from these mammalian species are similar in molecular charge and weight and are functionally similar in lecithin-cholesterol acyltransferase activation.

Characterisation of the alpha 1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 1. Protein staining.

The isoelectric points and the molecular weights of the major components of the eight Thoroughbred protease inhibitor (Pi) types have been determined by polyacrylamide gel isoelectric focusing and polyacrylamide gel pore gradient (ISO-DALT) electrophoresis respectively. The major Pi proteins focus in the range pH 3.74-4.43 and have molecular weights ranging from 55 000-72 000 daltons. Using the ISO-DALT method of electrophoresis, protein maps for the eight Thoroughbred Pi types have been presented for the first time. None of the homozygous Pi types are identical except for the types S1 and S2 which show partial identity. The results do not necessarily support Juneja et al.'s (1979) contention of two closely linked alpha 1 Pi systems based on molecular weight differences. It is suggested that the traditional nomenclature originally proposed by Braend (1970) be maintained to describe the eight Pi alleles in Thoroughbred horse plasma. The ISO-DALT method provides a sensitive technique which is superior to existing techniques for the analysis of the horse Pi system.

Characterisation of the α1‐protease inhibitor system in Thoroughbred horse plasma by horizontal two‐dimensional (ISO‐DALT) electrophoresis. 2. Protease inhibition

The protease inhibitory spectra of the eight homozygous Thoroughbred Pi types against trypsin, elastase and chymotrypsin have been determined. The alpha 1-protease inhibitor proteins exhibit three classes of inhibitory specificity towards these enzymes. The Pi types F, I, N and U exhibit class I (trypsin, elastase and chymotrypsin) and class II (trypsin and elastase) types of inhibition and fit Juneja et al.'s (1979) classification of two separate genetic systems Pi 1 and Pi 2 based on differences in the inhibitory spectra against trypsin and chymotrypsin. The remaining four Pi types are exceptions to Juneja et al.'s (1979) classification. Types G, L, S1 and S2 possess class I but not class II proteins. A third class of proteins (class III) which exclusively inhibit chymotrypsin was detected in all eight protease inhibitor types. Type G is well represented by class III proteins because two of the three major proteins of the ISO-DALT pattern inhibit only chymotrypsin and is thus an exception to Juneja et al.'s (1979) classification.