Pharmacokinetics and metabolism of rectally administered paracetamol in preterm neonates.

AIM: To investigate the pharmacokinetics, metabolism, and dose-response relation of a single rectal dose of paracetamol in preterm infants in two different age groups. METHODS: Preterm infants stratified by gestational age groups 28-32 weeks (group 1) and 32-36 weeks (group 2) undergoing painful procedures were included in this study. Pain was assessed using a modified facies pain score. RESULTS: Twenty one infants in group 1 and seven in group 2 were given a single rectal dose of 20 mg/kg body weight. Therapeutic concentrations were reached in 16/21 and 1/7 infants in groups 1 and 2, respectively. Peak serum concentrations were significantly higher in group 1. Median time to reach peak concentrations was similar in the two groups. As serum concentration was still in the therapeutic range for some infants in group 1, elimination half life (T1/2) could not be determined in all infants: T1/2 was 11.0 +/- 5.7 in 11 infants in group 1 and 4.8 +/- 1.2 hours in group 2. Urinary excretion was mainly as paracetamol sulphate. The glucuronide:sulphate ratio was 0.12 +/- 0.09 (group 1) and 0.28 +/- 0.35 (group 2). The pain score did not correlate with therapeutic concentrations. CONCLUSIONS: A 20 mg/kg single dose of paracetamol can be safely given to preterm infants in whom sulphation is the major pathway of excretion. Multiple doses in 28-32 week old neonates would require an interval of more than 8 hours to prevent progressively increasing serum concentrations.

Analysis of human tear fluid components, inhibiting protein adhesion to plastic surfaces.

In a previous paper we reported the presence of components in human tear fluid that block the interaction of proteins with plastic surfaces, interfering with tear protein ELISA and proposed the term coating inhibiting activity. The purpose of the study presented here was to further analyse these components. Coating inhibitory activity in human reflex tears was analysed by lectin affinity chromatography, using the agarose bound lectin Artocarpus integrifolia agglutinin (Jacalin), gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting and Jacalin staining. For coating inhibitory activity assay in experimental tear samples, the binding of the protein Avidin-conjugated horseradish peroxidase to the polystyrene surface of ELISA micro-titer plate wells, preincubated with the experimental tear samples was measured. In addition, tears were incubated with scrapings of the ELISA plates used in the assay and with six different types of contact lenses (two rigid gas permeable and four hydrogel soft contact lenses) for analysis of adsorbed components. Lectin affinity chromatography of tears yielded a Jacalin-binding and a non-Jacalin-binding preparation, both exhibiting coating inhibitory activity but representing chemically different preparations as observed by SDS-PAGE. After performing gel filtration, coating inhibitory activity eluted with similar retention in both preparations. In fractions exhibiting activity, tear proteins of low molecular weight (< 40 kDa) were detected. Among these, two Jacalin-binding glycoproteins were detected; a major component of approximately 28 kDa and a somewhat smaller minor component. All low molecular weight components were also detected on the scrapings, incubated with tears. The possibility that coating inhibitory activity in tears might reside in a component of larger molecular size can however not be excluded. The human tear proteins secretory Immunoglobulin A, lactoferrin and lysozyme are not involved in coating inhibition. On one of the two rigid gas permeable contact lenses incubated with the tears, the 28 kDa glycoprotein was detected. From the data obtained in our study we conclude that coating inhibitory activity in tears seems to be associated with multiple components of low molecular weight.

Analysis of tear fluid proteins in insulin-dependent diabetes mellitus.

Secretory immunoglobulin A, lactoferrin, lysozyme and tear specific pre-albumin were analyzed in stimulated tear fluid of 25 diabetic patients without retinopathy and in 29 diabetic patients with (pre) proliferative retinopathy using high performance liquid chromatography. Results were compared to those obtained in 26 healthy controls to determine the effect of diabetes mellitus on the exocrine function of the main lacrimal gland. Sodium dodecyl sulfate polyacrylamide gel electrophoresis onto minigels was performed on 20 tear samples for verification of high performance liquid chromatography fractions recorded. The mean total protein values in tear fluid (Bradford assay) of diabetics without retinopathy, with retinopathy and healthy controls did not differ significantly (mean in mg/ml +/- SD: 6.4 +/- 2.2, 5.9 +/- 2.0 and 5.7 +/- 1.7, respectively; Mann-Whitney; p > 0.02). High performance liquid chromatography showed an increased secretory immunoglobulin A and decreased peak 5 OD280 (+56% and -38%, respectively; p < 0.02) in patients without retinopathy, whereas in patients with retinopathy lysozyme was increased (+27%; p < 0.01) and tear specific pre-albumin and peak 5 OD280 decreased (-24% and -42%, respectively; p < 0.04), when compared to healthy controls. These inconsistent differences do not uniformly suggest an exocrine dysfunction of the main lacrimal gland in diabetic patients.

Identification of lectin binding proteins in human tears.

The identity of glycoproteins in stimulated normal human tears was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of tears onto minigels, blotting, and subsequent incubation with different biotinylated lectins (concanavalin A [Con A], peanut agglutinin [PNA], glycine max agglutinin [SBA], Phaseolus vulgaris agglutinin, wheat germ agglutinin [WGA, native form], Artocarpus integrifolia agglutinin [Jacalin], and Pisum sativum agglutinin). Control proteins included purified secretory immunoglobulin A (sIgA) from human colostrum, human milk lactoferrin, and chicken-egg lysozyme. All samples were prepared in a denaturing (SDS) buffer under nonreducing and reducing conditions. The sIgA in tears and IgA (alpha) heavy chain fragments (reduced sample) were identified with most of the lectins tested. A particular high molecular weight (greater than 200 kD) protein fraction in tears that just entered the separation gel on SDS-PAGE was detected with WGA and Jacalin. This fraction stain poorly with silver. Tear lactoferrin was identified with all lectins used, although binding was low with SBA. Purified milk lactoferrin showed a poor reaction with Jacalin, but a protein in tears of similar mobility bound this lectin (nonreduced samples). Under both nonreducing and reducing conditions, tear-specific prealbumin in tears did not bind any of the lectins tested. Tear lysozyme only reacted with lectin after reduction. The techniques described may provide additional valuable information in addition to commonly used methods for tear protein analysis and further knowledge concerning the role of glycoproteins on the ocular surface.

SDS-Minigel electrophoresis of human tears. Effect of sample treatment on protein patterns.

An automated Minigel electrophoresis system (PhastSystem, Pharmacia, Uppsala, Sweden) was tested for human tear protein analysis. Tear samples were treated under nonreducing or reducing conditions before sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE). Micro-amounts of tears (2 microliters) were sufficient for analysis and separation and visualization of proteins were completed within 2 hr. Tear proteins were identified using purified control proteins and immunoblotting techniques, using antisera against immunoglobulin (Ig) A (alpha) heavy chains, Ig heavy and light chains, secretory components and lactoferrin. In nonreduced tears, lactoferrin (seen as a double band), serum albumin, tear-specific prealbumin (TSPA), and lysozyme were clearly separated. Secretory IgA (sIgA) was seen as a smear on top of the gel. Immunostaining also showed a major Ig light chain containing protein. After reduction, the protein profiles showed marked changes. In reduced tears, immunoglobulin heavy and light chains (molecular weight [MW]: 64 and 28 kD, respectively) were detected on the SDS-PAGE profile after immunostaining, and represented disulfide cleavage fragments, which originated from sIgA. Reduction resulted in the liberation of the secretory component piece (MW: 85 kD), which was found to co-migrate with the tear lactoferrin bands. Both lactoferrin and serum albumin acted as larger proteins on SDS-PAGE after reduction. The authors found that the two methods of sample treatment, before electrophoresis, resulted in marked differences on the electropherograms.

Detection of secretory IgM in tears of IgA deficient individuals.

Tears from normal (n = 5) and serum IgA deficient (n = 3) individuals were investigated for the presence of secretory Immunoglobulin A (sIgA), sIgM and free secretory component (SC) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using 10-15% gradient minigels (PhastSystem), followed by immunoblotting using various immunological probes. Tear samples were treated in denaturing (SDS) sample buffer under non-reducing as well as reducing conditions, prior to analysis. All normal tear samples contained sIgA as well as free SC (estimated MW: 82kD) but only traces of IgM. Tears from the three serum IgA deficient subjects lacked sIgA but did contain free SC. In two of them sIgM was clearly detected and after treatment of tears with reducing agent, IgM (mu) heavy chain fragments (estimated MW: 78kD) were identified and could be distinguished from other tear proteins after SDS-PAGE. These findings indicate lacrimal secretion of free secretory component, even in the absence of its ligand. On the ocular surface, sIgM may play a compensatory role in IgA deficiency.

Sialic acid in human tear fluid.

A simple assay for the determination of sialic acid (N-acetylneuraminic acid) in human tear fluid was evaluated. Sialic acid, terminally bound on carbohydrate side-chains of glycoproteins, was released after treatment with neuraminidase and measured by an enzymatic colorimetric test. Tear fluid samples were collected from ten healthy adults, using glass capillaries and cellulose sponges. Sialic acid levels in tears collected with sponges (0.8-1.8 mmol l-1) did not differ significantly from those found in capillary tears (0.9-1.8 mmol l-1). Sialic acid, expressed as mmol g-1 protein, was significantly lower in tears collected with sponges (0.18-0.32 mmol g-1) than with capillaries (0.19-0.42 mmol g-1). Recovery of sialic acid and protein after incubation of cellulose sponges with tears was more than 99%. Sialic acid levels in human tears, which had been centrifuged to remove insoluble material, remained unchanged. Furthermore, tear sialic acid activity did not pass a filter with a molecular weight cut-off point of 10,000. Our data indicate that with the assay used in this report, sialic acid in tears is not due to secretory immunoglobulin A (sIgA), lactoferrin and lysozyme. The fact that the major tear proteins do not contribute to the sialic acid levels detected in tears suggests that other as yet unknown soluble glycoproteins are involved.

Gel electrophoresis of human tears reveals various forms of tear lactoferrin.

Lactoferrin is a metal binding protein, which is present in high concentrations in human tears. Little is known concerning the exact molecular shape of lactoferrin in tears. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting experiments showed that this protein is present in multiple forms in tear fluid. SDS-PAGE analysis of human tears under non reducing conditions and pretreatment of tears in sample buffer at room temperature revealed lactoferrin in a major form of 60 kD, a minor form of 64 kD and a third form of 52 kD. Pretreatment of tears at elevated temperatures prior to sample application resulted in the loss of this third form. Disruption of intrachain disulfide bridges prior to SDS-PAGE analysis resulted in a shift in the apparent molecular weight of lactoferrin to 78 kD and 83 kD for the major and minor form, respectively. Chromatography of human tears on ConA-Sepharose as well as enzymatic deglycosylation showed that the difference in molecular weight of the major and minor lactoferrin form was not due to a variation in the carbohydrate side chains. The presence of the minor form could also not be ascribed to iron saturation. Instead we found that addition of iron ions to human tears resulted in a shift of tear lactoferrin to a lower molecular weight species of about 52 kD, coinciding with the third lactoferrin form mentioned above and a small protein band of approximately 57 kD, representing the iron saturated minor lactoferrin form. Similar findings were observed using purified milk lactoferrin. Increasing the temperature prior to sample application or disruption of disulfide bridges dissociated the iron-lactoferrin complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin F2 alpha isopropyl ester versus iloprost phenacyl ester in rabbit and beagle eyes.

One and 2 micrograms of prostaglandin F2 alpha isopropyl ester (PGF2 alpha -IE) applied topically to the rabbit eye caused a biphasic response. The hypotensive phase was dose-dependent with a maximum reduction in intraocular pressure (IOP) of 9.4 +/- 1.7 mmHg at a dose of 2 micrograms. In beagles, 0.4 to 2 micrograms topical PGF2 alpha-IE resulted in a sustained IOP reduction; 2 micrograms produced the maximum reduction of 7-9 mmHg. No initial hypertensive response was observed. Iloprost phenacyl ester (Iloprost-PE) caused a greater decrease in IOP when dissolved in 0.5% hydroxypropyl methylcellulose (AT) than in saline. In rabbits, doses of 0.1 to 1 microgram in AT caused a biphasic response with a sustained IOP decrease fluctuating between 7 and 8 mmHg. In beagles 1 and 2 micrograms Iloprost-PE resulted in a mean IOP reduction of 5.8 +/- 0.4 mmHg and 5.8 +/- 0.5 mmHg (P less than 0.005), respectively; the decrease persisted for 5 hrs. No initial hypertensive response was observed. In beagles PGF2 alpha-IE induced a strong miosis lasting more than 6 hours; Iloprost-PE had no effect on pupil size. Both PG-esters induced a slight hyperemia in rabbit and beagle eyes. In rabbits Iloprost-PE affects the blood-aqueous barrier more than PGF2 alpha-IE, since higher protein concentrations are seen in the aqueous humor after application of Iloprost-PE. Neither PG-ester had a noticeable effect on aqueous protein in beagles. In rabbits, both PG-esters led to slightly increased aqueous humor cyclic-AMP concentrations. In beagles aqueous humor cyclic-AMP was elevated only after Iloprost-PE.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors influencing the quantitative determination of tear proteins by high performance liquid chromatography.

HPLC analysis of human tears allows tear protein profiles to be obtained within ten minutes. A tear protein profile normally consists of 4 major peaks: IgA, lactoferrin, protein G and lysozyme. Although it is a rapid method, the use of High Performance Liquid Chromatography in the (quantitative) determination of proteins in tears is influenced by various factors. The day to day variability of the quantitative use, ranges between 7 and 9% for the various tear proteins. Combining the HPLC method with a convenient collection method such as sponges or Schirmer strips, showed that the sponges and some of the Schirmer strips used in this study eluted significant material absorbing light at 280 nm. No statistical difference was observed in the HPLC protein profiles of tears collected with Schirmer strips or with sponges. Using sponges has the advantage that they can absorb almost twice as much tears in a same period of time as Schirmer strips. HPLC analysis of human tear proteins is not accurate when there is albumin leakage as in traumatic sampling with Schirmer strips or in inflammatory states. The I-125 column which was used in our study is not able to separate lactoferrin and albumin, which may cause an overestimation of lactoferrin in inflammatory conditions. The study presented here indicates that for quantitative use of HPLC in epidemiological tear protein research better separating protein gel filtration columns are needed.

Inhibition of hydroxyl radical formation by human tears.

The effect of human tears on oxygen radical formation was investigated using xanthine-xanthine oxidase as the oxygen radical generating system. Superoxide (O2.-) and hydrogen peroxide (H2O2) were measured using ferricytochrome c as indicator. OH. formation was monitored by measuring the hydroxylation of salicylate. Addition of traces of iron (Fe3+) and chelator (EDTA) was a prerequisite for OH. formation in this system. Human tears did not detectably affect O2.- or H2O2 formation but markedly inhibited OH. formation. Tears obtained from eight different individuals all showed a marked inhibitory effect on OH. formation, whereby only a small individual variation was observed. During separation of human tears by gelfiltration on a Sephadex G75 column, three protein peaks eluted from the column. The first contained lactoferrin, the second as yet unidentified material, and the third lysozyme. Inhibitory activity on OH. formation coincided with the first protein peak and also with fractions eluting after the protein peak containing lysozyme. The major inhibition on OH. formation was seen in these latter fractions, which contain small organic and anorganic substances. The fact that ascorbic acid could not be detected in human tears and that it did not affect formation of OH. in this investigation's assay system indicates that this compound was not involved in the observed low molecular weight inhibitory effect. Analysis of various cations suggested that the low molecular weight inhibitory effect could largely be ascribed to tear calcium. Tear calcium binds to EDTA and thus possibly prevents formation of the essential catalytic iron-EDTA complex. Experiments using purified human milk lactoferrin showed, that this protein, which is abundantly present in human tears, can inhibit OH. formation in the model used here. The inhibitory effect of lactoferrin was counteracted by increasing the iron concentration in the reaction mixtures. These findings suggest that tear lactoferrin may play an important role in the protection of the ocular surface against OH. induced damage.