Impact of 6% hydroxyethyl starch 130/0.42 and 4% gelatin on renal function in a pediatric animal model.

OBJECTIVES: Artificial colloids, frequently used to prevent hemorrhagic shock in children, may induce serious renal side effects in critically ill adult patients. The impact of perioperative colloid infusion on the renal function in adults and children remains unclear. AIM: To determine the impact of single doses of artificial colloids on renal function tests, we conducted an experimental animal study. We hypothesized that neither the infusion of moderate doses of 6% hydroxyethyl starch (HES) nor of 4% gelatin (GEL) would have a serious impact on the renal function of healthy piglets. METHODS: Fifteen sedated piglets were randomly assigned to receive an infusion of either 20 ml·kg(-1) HES or GEL or a balanced electrolyte solution (BS, control group) over 30 min. Before and 7 days after infusion, serum and urine renal function tests were recorded and renal biopsies were taken. RESULTS: Serum and urine renal function tests (e.g., creatinine, urea, cystatin C, and neutrophil gelatinase-associated lipocalin) were within normal ranges, and a microscopic examination of the renal tissue in all groups revealed no major alterations such as tubular necrosis, interstitial bleeding, interstitial inflammation, or vacuoles. CONCLUSIONS: In this pediatric animal model, the infusion of moderate doses of artificial colloids was not found to have any relevant impact on renal function. Further clinical investigations are necessary to provide a conclusive assessment of the risk for renal impairment after HES and GEL administration during major pediatric surgery.

A rapid analytical method for the detection of plasma volume expanders and mannitol based on the urinary saccharides and polyalcohols profile.

A screening procedure specifically developed for the detection of saccharides and polyalcohols in human urine in the framework of doping control analysis is presented. The proposed method, set-up, and validated to detect the abuse of dextran, hydroxyethyl starch and mannitol as a doping practice in sport, involves only one enzymatic hydrolysis step and the direct injection into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The chromatographic conditions were optimized to allow the efficient separation of compounds with the same molecular weight. Good linearity (R(2) 0.990-0.995) and reproducibility of relative retention times (CV% lower than 1) and of relative abundances of characteristic ion transitions (CV% lower than 10) were obtained. The lower limits of detection and quantification were in the range of 30-100 µg/ml. Since the analytes studied are present also in non-doping products (e.g. in fruit as well as in food products and drugs additives), the developed method was also used to establish a range of reference urinary concentrations: 600 doping control samples and 30 samples from volunteers not using any medication were considered. While the hydrolysis products (isomaltose and maltose hydroxyl-ethylated), used as specific markers for the detection of dextran and hydroxyethyl starch abuse, were not detected in urine; mannitol was present in all urines in a concentration range of 30-1200 µg/ml. Since no criteria of positivity for mannitol has been established yet, the results obtained in this study could be considered, in combination with those of previous researches, as a starting point to fix a threshold value for doping control purpose.

Specific screening method for dextran and hydroxyethyl starch in human urine by size exclusion chromatography-in-source collision-induced dissociation-time-of-flight mass spectrometry.

The use of plasma volume expanders (PVE), such as dextran (DEX) and hydroxyethyl starch (HES), is prohibited in sports. DEX is a naturally occurring glucose polymer, whereas HES is synthetically produced from amylopectin starch by substitution with hydroxyethyl groups. In doping control, the commonly applied enzymatic and colorimetric screening methods are lacking adequate specificity for DEX and HES. Also, gas chromatographic-mass spectrometic (GC-MS) screening methods have specificity issues with DEX. In addition, due to the nature of the target compounds, time-consuming derivatisation steps are required in GC-MS. Based on the high molecular weight of carbohydrate polymers excreted in urine after administration of DEX and HES, a screening method was developed involving size exclusion chromatography (SEC) combined with time-of-flight mass spectrometry (TOFMS). By using solely a SEC guard column as an analytical column allowed sufficient chromatographic resolution in a minimal amount of time and with reasonable repeatability (average RSD of 10%). Detector response was linear throughout the measurement range with R(2) > 0.99 for both analytes. Limits of detection were 100 and 250 µg mL(-1) for DEX and HES, respectively. Ion suppression was found to be 52% at maximum. In-source collision-induced dissociation (ISCID) was used to produce characteristic fragments at a mass accuracy better than 2 mDa. The specificity of the SEC-ISCID-TOFMS method was demonstrated with 120 PVE negative doping control samples analyzed in parallel with a routine GC-MS screening method. In addition, seven urine samples from diabetic athletes, causing interpretation problems in routine GC-MS, showed here a definitely negative profile.

Microwave irradiation for a fast gas chromatography-mass spectrometric analysis of polysaccharide-based plasma volume expanders in human urine.

In this contribution we tested the possibility to use microwave irradiation for the screening and confirmation pre-treatment steps of hydroxyethylstarch, with the aim to speed up gas chromatography-mass spectrometric procedures. Acid hydrolysis and derivatization processes were conducted in a temperature-controlled single beam microwave oven for organic synthesis. The kinetics of hydroxyethylstarch chemical hydrolysis and derivatization were investigated at different microwave power, incubation temperature and incubation time. The best hydrolysis conditions were found at a microwave power value of 1200 W (T 100°C) with an incubation time of 2 min; whereas the best derivatization conditions were found at a microwave power value of 1020 W (T 100°C) with an incubation time of 5 min. The effectiveness of this approach was evaluated by gas chromatography-mass spectrometry analyzing more than 20 different pools of blank urine samples spiked with hydroxyethylstarch at a concentration of 1 mg/mL. The results showed that the effect of microwave irradiation on the chemical hydrolysis process was very remarkable: the total sample preparation time can be shortened by 58 min compared to the reference method (2 min instead of 60 min). In addition to this, the time necessary for the derivatization process can also be drastically shortened with respect to the reference procedure (5 min instead of 30 min). The repeatability of the hydrolysis and derivatization recoveries, the limit of detection and the matrix interferences were comparable to the reference method accredited under the ISO 17025 guidelines and presently followed by the accredited sports anti-doping laboratory of Rome.

Screening for hydroxyethyl starch (HES) doping in sport.

The artificial colloid hydroxyethyl starch (HES) is among the most frequently used plasma volume expanders in the medical field. However, in 1998, its misuse by the athletic community was officially reported and since 2000, HES is prohibited by the International Olympic Committee (IOC). Therefore, several methods enabling the detection of HES in urine were developed, most based on gas chromatography-mass spectrometry (GC-MS). In the present work, a simple and low-cost screening method, intended to reduce the number of samples to be analysed by GC-MS, was developed. The method is based on the acid hydrolysis of HES and detection of the resulting glucose and hydroxyethyl glucose derivatives by Benedict's reaction (reduction of copper sulfate to brick-red cuprous oxide by glucose and/or derivatives). Samples considered suspect were submitted to GC-MS analysis for identification of HES. The method was successfully applied for screening of HES in 2627 urine samples from 1346 Brazilian soccer players and 1281 athletes from the Pan-American Games (Rio de Janeiro, 2007); 71 (2.7%) samples, considered suspect, were submitted to GC-MS, but no positive results were found. Moreover, a thin layer chromatography (TLC) method was adapted for visualisation of the characteristic band pattern of HES hydrolysis products. The results indicate that the methods are efficient and useful for the screening of HES in urine.

Rapid screening of polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch in human urine by liquid chromatography-tandem mass spectrometry.

The increasing number of samples and target substances in doping control requires continuously improved screening methods, combining high-throughput analysis, simplified sample preparation, robustness and reliability. Hence, a rapid screening procedure based on liquid chromatography-electrospray ionization-tandem mass spectrometry with in-source collision-induced dissociation was developed. The detection of the polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch (HES) in human urine was established without further sample preparation. The in-source fragmentation strategy of the approach represented a valuable tool in the analysis of the polysaccharide-based compounds, allowing the use of tandem mass spectrometry. After direct injection of urine specimens, analytes were chromatographically separated on a monolithic reverse-phase column and detected via multiple reaction monitoring of diagnostic ions at detection limits of 10 microg/mL for HES and 30 microg/mL for dextran. Validation was performed regarding the parameters specificity, linearity, precision (8-18%) and accuracy (77-105%) and the method was applied to the investigation of approximately 400 doping control samples and seven dextran and two hydroxyethyl starch post-administration samples. The approach demonstrated its capability as a rapid screening tool for the detection of dextran and hydroxyethyl starch and represents an alternative to existing screening procedures since time consuming hydrolysis or derivatization steps were omitted.

Detection of hydroxyethylstarch (HES) in human urine by liquid chromatography-mass spectrometry.

The objective of this study was to establish the possibility of using liquid chromatography coupled to mass spectrometry for the detection of hydroxyethylstarch (a corn starch derived product) in urine as an alternative to the current time consuming GC-MS methods. Analyses were performed using an ion trap instrument after acidic hydrolysis. Ionization was carried out using atmospheric pressure chemical ionisation (APCI) operated in negative ionization mode and detection was performed using MS(2). The results indicate that the developed method can successfully be applied as a fast and reliable method for the detection and identification of hydroxyethylstarch.

Rapid screening of plasma volume expanders in urine using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

The use of plasma volume expanders, especially those based on chemically modified polysaccharides such as hydroxyethyl starch, has found its way from the medical field to the athletic community in the everlasting drive for performance enhancement. As such, plasma volume expanders have been placed on the list of banned substances by the International Olympic Committee, and in turn require accurate and sensitive analytical tools for their detection in complex biological matrices. Here we present a relatively straightforward method for the detection of polysaccharide-based plasma volume expanders (PVE) in urine, based on the carefully controlled partial acid hydrolysis of urine (20 microL) in a total volume of 500 microL 4 M trifluoroacetic acid. Following the incubation (30 min at 100 degrees C) an aliquot of the hydrolysate is dried, re-suspended in the analytical matrix (e.g. 2,5-dihydroxybenzoic acid) and examined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). The obtained mass spectrometric profile reveals a high number of characteristic peaks in the mass range between 500 and 3000 Da, a region that in urine samples devoid of PVE appears relatively clean, and thus allows the unambiguous identification of the presence of such PVE. This approach is fast (the mass profile can be obtained within 90 min), highly sensitive (the effective sample amount on the MALDI target is equivalent to 100 nL urine), needs little sample handling (four steps), requires no derivatisation and is devoid of interference from other biomolecules. The approach has been worked-out for hydroxy ethyl starch but can be applied to other polymer-derived plasma expanders such as dextran and probably the newly developed acetyl starch.

Detection of the plasma volume expander hydroxyethyl starch in human urine.

The plasma volume expander hydroxyethyl starch (HES) is usually administered in cases of hypovolaemic shocks but in 1998 the press reported its misuse in endurance sports. Since January 2000, it has been put on the list of prohibited substances of the International Olympic Committee (IOC) and its misuse is to ban by doping controls. Therefore, a rapid method enabling the screening for HES in human urine was developed which can be easily adopted by IOC laboratories to analyse routine urine samples for this remedy. Excretion study urine samples obtained from patients treated with HES, blank urine specimen and reference standards, were hydrolysed with hydrochloric acid and without any further purification of the resulting monosaccharides their per-timethylsilylated derivatives were performed. By means of gas chromatography-mass spectrometry the products were separated and the alpha- and beta-isomers of glucose, 2-, 3- and 6-hydroxyethyl glucose derivatives were identified. Typical ion traces of 2- and 3-substituted glucose (m/z 248, m/z 261 and m/z 235, m/z 248, respectively) support the fast determination of the substances whose electron impact mass spectra are presented and discussed.

Intestinal elimination of hydroxyethyl starch?

OBJECTIVE: Hydroxyethyl starch (HES) is mainly eliminated via the kidneys. Any information about extrarenal elimination obtained so far has been either incomplete or contradictory. The objective of this study was to quantify the intestinal excretion of infused HES with a mean molecular weight of 200,000 and a molar substitution of 0.5 (HES 200/0.5) and to compare the reappearance/recovery rate in urine and plasma. DESIGN: Prospective clinical study without control group. SETTING: The study was conducted at the Institute of Hypertension of the Society of Clinical Pharmacology, Vienna, Austria, which is an establishment for research in volunteers. PARTICIPANTS: The results of six out of seven healthy male volunteers were appropriate for analysis. One trial subject had to be excluded from the study because of severe protocol violation (mixing of stool and urine samples). INTERVENTIONS AND METHODS: Each volunteer was administered 500 ml of 10% HES 200/0.5 in a 0.9% NaCl solution intravenously within 1 h. A gut lavage with 6 l of a polysaccharide free solution was continuously administered from 3 h prior to until 2 h after the HES infusion to facilitate the collection of the samples and to exclude any source of error at analysis. HES was quantified with the hexokinase method. MEASUREMENTS AND RESULTS: Right from the beginning of the infusion until 10 h after its completion, the cumulative HES excretion with feces (principle parameter) and urine as well as selective plasma volume and HES plasma level were measured. Six and 14 h after the infusion had been completed, the recovery rates of HES in urine were about 30% and 40%, respectively, and in plasma about 23% and 8%, respectively. By contrast, not more than a kind of "background noise amount" of HES (about 0.2 %) could be recovered in feces ( mean value in % of the infused amount of the substance). Six and 14 h after the infusion had been completed, the total recovery rates of HES were 53% and 49%, respectively. CONCLUSION: In a physiologically unimpaired gut HES 200/0.5 is not, or only to an infinitesimal extent, eliminated via the intestine. The question if there is any alternative path to renal excretion for HES still remains to be answered. As the calculated reappearance/recovery rate of HES is only about 50 % of the administered dose, further investigations as to the final fate of HES appear necessary.

Studies on hydroxyethyl starch. Part II: Changes of the molecular weight distribution for hydroxyethyl starch types 450/0.7, 450/0.5, 450/0.3, 300/0.4, 200/0.7, 200/0.5, 200/0.3 and 200/0.1 after infusion in serum and urine of volunteers.

32 volunteers, none of whom showed any symptoms for kidney, liver or pancreas disease, were given by infusion 500 ml of various type of hydroxyethyl starch (HES) at a concentration of 6% (450/0.7, 450/0.5, 450/0.3, 300/0.4) as well as of 10% (200/0.7, 200/0.5, 200/0.3, 200/0.1) over a period of 30 min. After infusion both the Mw and the Mn diminished. The rate of elimination of HES from serum entirely depended on molar substitution and not on Mw. The quotient Mw/Mn decreased considerably over the entire test period. The lower molecular weight limit in serum remained relatively the same at 60,000 Daltons. Maximum molecular weight limit of urine, too, was 60,000 Daltons.

Hydroxyethyl starch serum levels in leukapheresis donors measured by modified periodic acid-Schiff staining technique.

Hydroxyethyl starch (HES), a glucopyranose polymer used to enhance the yield of granulocytes during leukapheresis, was detected by periodic acid-Schiff (PAS) staining of 1-microliter samples following electrophoresis on thin agarose film. HES migrated with restricted electrophoretic mobility in the far gamma region, and the intensity of staining was found to be concentration dependent. Densitometer scanning showed a linear curve from 30 mg to 600 mg per dl, although bands were identified at concentrations lower than 30 mg per dl. Sera from 46 leukapheresis subjects showed the same restricted bands. HES measured retrospectively in sera obtained on days 1 to 400 following apheresis ranged from 675 to 30 mg per dl. The clearance of HES from the sera of four donors measured serially appeared biphasic with the first T1/2 of 0.4 to 6.4 days and the second T1/2 of 58 to 240 days. HES also was detectable in urine concentrated 100fold for as long as 7 days following leukapheresis . This simplified, and sensitive PAS glycoprotein staining method can be used to detect and follow HES concentrations in serum and may provide a new tool for the study of HES in leukapheresis donors.

[Evaluation of colloidal volume replacement solution 6% HES 40/0.5]. / Zur Bewertung des kolloidalen Volumenersatzmittels 6% HAS 40/0,5.

We studied the effect of a 6% hydroxyethyl starch 40/0.5-solution on circulating blood volume, blood composition, blood pressure and urine excretion in 20 healthy volunteers, suffering from moderate hypovolemia. In 10 cases we controlled by the Anthron method: Hydroxyethyl starch in serum and hydroxyethyl starch in urine. The hypovolemia was managed by collecting 400 ml blood from each volunteer. Afterwards 500 ml of the test solution were infused within 15 min. We measured the control parameters before and after the collection of 500 ml blood as well as 5, 90 and 240 min after the infusion of the test solution. At 5, 90 and 240 min after infusion time of the test solution the intravascular volume effect was reported at 71%, 103%, and 91% of the infused amount. After the infusion the systolic and diastolic pressure remained unchanged, while a decrease in pulse rate, a slight increase of glucose concentration in both urine and serum were reported. Decreases in values after the infusion were registered according to the dilution of blood for: serum protein concentration, hemoglobin concentration, erythrocytes, hematocrit. During the entire observation period osmolarity and colloid osmotic pressure in serum underwent no demonstrable changes. An increase in urine excretion was observed in the sense of an osmotic diuresis after the infusion. The calculated value of the rest fraction (the portion of the infused amount of hydroxyethyl starch, which was neither excreted in urine nor found in serum) was less than 2% of the infused substance, 240 minutes after the infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary excretion kinetics of hydroxyethyl starch 350/0.60 in normovolaemic man.

Rates of urinary excretion concomitant with the molecular size distribution of filtered polymer fragments were determined in five normovolaemic men dosed with 30 g/m2 BSA of a species of HES (HES 350/0.60) possessing a M-W of 350 000 combined with an MS of 0.60. Approximately 13% of the total injected dose of HES 350/0.60 was excreted in urine 1 hr after dosing, and 45% by 72 hours. Gel filtration of Sepharose CL-4B revealed that aliquots of urine collected 1 hour after injection contained polymer fragments of HES 350/0.60 with values of Kav ranging between 0.88 and 0.84, and possessed a Stokes radius (r = 32A) similar to that of Dextran 20 (M-W 22 700). Polymer fragments of HES 350/0.60 excreted 6 to 48 hours after dosing, however, possessed a Kav ranging between 0.78 and 0.73 with a Stokes radius (r = 45A) similar to that of Dextran 40 (M-W 41 000). All filtered polymer fragments were less polydisperse relative to both the injection solution (Kav 0.60) and residual material recovered from blood immediately after injection (Kav 0.72). These data support the hypothesis that the excretion of HES 350/0.60 occurs in two distinct phases: a rapid phase of elimination dependent on the M-n of the injected solution, and a slower phase dependent on the MS (degree of resistance to alpha-amylase attack). This study, in conjunction with our previous investigation of the changes in circulating HES 350/0.60, define the basic differences between clearance and excretion of the dextrans and of the rapidly degraded species of HES. These data are relevant to the utilisation of HES 350/0.60 during centrifugal leucapheresis.

[Effect of kidney function on the elimination and action of colloidal plasma substitutes]. / Einfluss der Nierenfunktion auf die Elimination und Wirkung von kolloidalen Plasma-Ersatzmitteln.

Hypovolemia and prophylaxis of thrombosis are the main indications for colloidal plasma substitutes. This is also true for patients with renal failure. Due to their difference in molecular structure colloidal plasma substitutes, which are in clinical use--dextran, hydroxyethyl starch, gelatin--, are eliminated tbrough different pathways. However, the kidney plays an important role in the elimination of all three substances. Therefore pharmacokinetics and pharmacodynamics of colloidal plasma substitutes, which are based on dextran, hydroxyethyl starch and gelatin, were studied in patients with normal or impaired renal function. Renal insufficiency proved to affect the elimination and therapeutic efficacy of these colloids in different and characteristic ways. Consequently this requires special indications and dosage recommendations for patients with renal insufficiency.

[Intravascular persistence, tissue storage and excretion of hydroxyethyl starch (HAS)]. / Intravasale Persistenz, Gewebsspeicherung und Ausscheidung von Hydroxyäthylstärke (HAS).

Hydroxyethyl starch is a derivative of amylopectin, the branched glycogen-like alpha-1,4-glucose polymer, the amylase hydrolysis of which is retarded by hydroxyethylation. If 70 to 90 per cent of the glucose polymer units contain hydroxyethyl groups, the intravascular persistence and urinary excretion of hydroxyethyl starch of a molecular weight of 435,000 is similar to that of Dextran 70. Hydroxyethyl starch and Dextran 70 are stored briefly in reticuloendothelial and hepatic cells, and cause swelling and vacuolation of renal tubules with little alteration of renal function. Elimination of hydroxyethyl starch from sites of tissue storage is much faster than elimination of the non-metabolized polymers acacia and polyvinylpyrrolidone. The distribution and excretion kinetics of hydroxyethyl starch are thus appropriate for a plasma substitute.

Transfusion of hydroxyethylated amylopectin-protected frozen blood in man. I. Plasma clearance and renal excretion of the cryoprotectant.

In man following the autologous transfusion of blood previously frozen with 14% low molecular weight-hydroxyethylated amylopectin (cryo-HES), the clearance of this material from the intravascular space was compound, and appeared to consist of exponential components. The overall half-life -- however, was 10.6 +/- 3.0 (SD) h. Approximately 17% of the total infused cryo-HES was excreted in the urine 1 h postinjection, and 40% by 72 h. The erythrocyte sedimentation rate (ESR) was not affected by the presence of this substance in the bloodstream of the recipient. The results indicate that cryo-HES is removed rapidly following the transfusion of blood previously frozen with this material.