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Hydroxyethyl Starch: The Effect of Molecular Weight and Degree of Substitution on Intravascular Retention In Vivo

Hitosugi, Takashi DDS*; Saito, Toshiyuki MD, PhD*†; Suzuki, Sono DDS*; Kubota, Ieko DDS*; Shoda, Emi DDS*; Shimizu, Toru MD, PhD; Oi, Yoshiyuki MD, PhD*

doi: 10.1213/
Critical Care and Trauma: Research Report

BACKGROUND: Hydroxyethyl starch (HES) solution is characterized by its mean molecular weight (MW), concentration, and degree of substitution (DS). This character varies worldwide.

METHODS: After binding fluorescein-isothiocyanate (FITC-HES), we evaluated the retention rate of three types of 6% HES in the A2 and V2 blood vessels of rat cremaster muscles using intravital microscopy in a mild hemorrhage model (10% of total blood volume). After blood withdrawal, we infused three types of FITC-HES: HES-A (MW 150–200 kDa, DS 0.6–0.68), HES-B (MW 175–225 kDa, DS 0.45–0.55), or HES-C (MW 550–850 kDa, DS 0.7–0.8) before determining the FITC-HES retention rate in the intravital microscope.

RESULTS: For V2, the FITC-HES retention rates 120 min after the start of the infusion were 27% ± 7.2% of baseline values (HES-A), 65% ± 9.1% (HES-B), and 86% ± 9.6% (HES-C); for A2 they were 27% ± 6.6%, 73% ± 10.2%, and 89% ± 8.7%, respectively. HES-B and HES-C were retained in the vessels longer than HES-A (P = 0.028 for V2, P = 0.038 for A2 between HES-B and HES-A; P = 0.022 for V2, P = 0.037 for A2 between HES-C and HES-A). There was no difference in the rate of disappearance from the vessels between HES-B and HES-C.

CONCLUSIONS: HES-B and HES-C are equally retained in the blood vessels. Middle-sized HES-B with low DS and middle substitution pattern stayed in the blood vessels as long as the large-sized HES. HES solutions of varying characters should be examined to optimize HES infusion.

IMPLICATIONS: Both the molecular weight and the degree of substitution of hydroxyethyl starches modify their retention within the circulation. The properties of individual hydroxyethyl starch solutions need to be further characterized.

From the *Department of Anesthesiology, Nihon University Graduate School of Dentistry; †Department of Anatomy, Tokyo Medical University, Tokyo, Japan; and ‡Department of Bioengineering, University of California, San Diego, La Jolla, California.

Accepted for publication May 14, 2007.

Supported by a Nihon University Individual Research Grant (Y.O.); Research Grants from the Uemura Fund (Y.O.), the Sato Fund (Y.O.), and the Dental Research Center (Y.O.), Nihon University School of Dentistry; the Promotion and Mutual Aid Corporation for Private Schools of Japan (Y.O., T.H.); and a grant-in-aid for scientific research (17592112) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Y.O.).

Presented in part at the International Symposium on Intensive Care and Emergency Medicine 2006, Brussels, Belgium.

Address correspondence and reprint requests to Yoshiyuki Oi, MD, PhD, Department of Anesthesiology, Nihon University Graduate School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan. Address e-mail to

Crystalloid and colloid solutions are generally used as intravascular volume expanders for hemorrhage in the surgical theater (1,2). There are hypotonic, isotonic, and hypertonic crystalloids, and human albumin, dextran, gelatin, and hydroxyethyl starch (HES) in colloid solutions. Each HES solution is characterized by its molecular weight (MW), concentration, degree of substitution (DS), and the substitution pattern ratio (C2/C6) (3). The pharmacological effects of HES depend on the number of starch particles in the vascular space, which produce the colloid osmotic pressure. Therefore, it is helpful to observe the leakage of the HES through the vascular wall to assess the retention of HES.

To optimize the use of HES and to characterize the ideal HES, we need to know how long it actually stays in the blood vessels. This study evaluated different sizes and molecular structures of HES bound with fluorescein-isothiocyanate (FITC-HES). We used intravital microscopy with a spectrophotometer-computer detection system to determine the rate of leakage of the HES from the microvessels and thus to examine the retention of HES particles.

Rat cremaster muscle was chosen to observe the vessels distributed over an area of muscular fascia (4–9). A model of mild hemorrhage, which occurs frequently during surgery, was chosen for this study. This model allowed us to optimize animal survival and to study the response of animals to the hemorrhage over 2 h.

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The study was approved by the Committee for Ethical Review of Animal Experiments at Nihon University, and was conducted according to the National Institutes of Health guidelines for the care and use of experimental animals.

We produced FITC-HES as described by Thomas et al. (10) from noncommercial HES for experimental use (Ajinomoto Pharma Co., Ltd., Japan). We used three types of HES with different characters: HES-A (MW 150–200 kDa, DS 0.6–0.68, C2/C6 = 8:1), HES-B (MW 175–225 kDa, DS 0.45–0.55, C2/C6 = 6:1), and HES-C (MW 550–850 kDa, DS 0.7–0.8, C2/C6 = 5:1). The volume of distribution of FITC-HES was extrapolated to time zero of the natural logarithms of the fluorescence derived from plasma samples obtained at the three time points.

Male Sprague-Dawley rats (5 ± 1 wk old, weighing 200 ± 10 g) were purchased from Sankyo Laboratory Co. Ltd. (Tokyo, Japan). The rats were maintained under controlled conditions at a temperature of 23°C ± 3°C with a relative humidity of 55% ± 15% and a 12:12 h light–dark cycle. They were used for experiments after acclimatization of at least 14 days to these conditions, and fasted for 24 h before the experiments, but had free access to water.

Anesthesia was induced with intraperitoneal sodium pentobarbital (1 g/kg). A venous line was taken at the ventral tail vein with a 24-gauge needle to provide continuing anesthesia with pentobarbital (40–90 mg · kg−1 · h−1). Normal saline was administered as the maintenance fluid at a rate of 2.5–5 mL · kg−1 · h−1. The infusion rate was adjusted to maintain a stable light level of anesthesia based on the previous criteria (11). Animals were placed on a warming pad to maintain body temperature between 37°C and 38°C; temperature was monitored by a rectal thermometer. The trachea was intubated with a polyethylene catheter (internal diameter: 2.1 mm) to support spontaneous breathing of room air.

A silicone cannula was inserted into the left carotid artery to allow continuous monitoring of arterial blood pressure via a pressure transducer (BP-308ETI, OMRON Corporation, Tokyo, Japan). The femoral artery was catheterized for blood withdrawal. For the observation of intravital microscopy, the cremaster muscle was prepared (6). When the anesthetized rats were lying with the cremaster flat on the microscope slide, they were allocated to one of the three experimental groups: re-infusion with HES-A, re-infusion with HES-B, or re-infusion with HES-C, all in saline solution.

In all groups, under anesthesia, hemorrhage was induced by removing 10% of the total body blood volume at a constant rate over a 10-min period (approximately 0.1 mL · min−1 · 100 g−1). A 30-min stabilization period was followed. During this period, areas of measurement were selected randomly to assess microcirculatory variables, and four points were fixed for observation. The first two points were the points 100 μm from the A2 and V2 wall to the outside, classified according to their order of branching, as previously described (5). The other two points were inside A2 and V2.

At t = 0 min, 0.3 mL/100 g FITC-HES was administered systemically via the tail vein. The hemorrhage and stabilization periods were followed by a constant infusion of one of the three types of FITC-HES at the rate of 0.1 mL · min−1 · 100 g−1. The brightness of the illumination at the four points was measured for a 30-min baseline period and for an experimental period of 120 min (t = 0–120 min) in all groups. The retention of HES was evaluated by the contrasting density of the brightness of fluorescence on the image stored in a personal computer.

For the observation of the intravital fluorescent microscopy, the animal on the warming pad was transferred to the modified stage of a BX51WI Olympus fluorescent microscope (Olympus Co., Ltd., Tokyo, Japan). This was equipped with a tungsten lamp for transmitted light and a mercury arc lamp for epi-illumination fluorescent light microscopy. A filter cube blocked the path of epi-illumination of the cremaster microcirculation. The extravasation of FITC-HES was measured by determining the changes in integrated optical intensity by image analysis.

The FITC-HES represented relative changes in retention and permeability, as previously described (8). In brief, ΔI = 1 – (IiIo)/Ii, where ΔI is the change in light intensity; Ii is the light intensity inside the vessel, and Io is the light intensity outside the vessel. Each experimental frame was digitized into a 512 × 512 charge-coupled device (DP70; Olympus Co., Ltd.).

FITC-HES was infused through the tail vein into the circulating blood. FITC-HES is normally retained in vasculature; therefore, epi-illumination allows the cremaster muscle microcirculation to be clearly visualized (2). Images of the preparation were monitored using a charge-coupled device camera (DP70; Olympus Co., Ltd.), displayed on a high-resolution monitor (Olympus Co., Ltd.), and saved to a hard disk drive (Olympus Co., Ltd.) for later off-line analysis.

Four areas of cremaster muscle microcirculation (random location) were recorded by using epi-illumination to include two A2 (30–70 μm) arterioles and two V2 (35–120 μm) venules in each animal (6). FITC-HES leakage from the A2 and V2 vessels was recorded every 5 min and assessed offline using an Olympus personal computer (Olympus Co., Ltd.) and an image analysis software package (MetaMorph®, Molecular Devices, Sunnyvale, CA). Each of the 3–5 areas selected was intermittently exposed to fluorescent light for a maximum of 15 s at each 5-min interval to produce photodynamic effects (2). Macromolecular leakage was assessed based on the contrasting density of the brightness of FITC-HES fluorescence on the image stored on a computer. Permeability was measured by light intensity taken at two different sites, one within the vessel and the other adjacent to the vessel, within the same area.

All data are expressed as mean ± se. One-way analysis of variance was used to compare the means of different treatments. If significance was identified, individual comparisons were subsequently made using the Scheff é t-test to determine the site of significance within the data sets. The differences were considered significant when P < 0.05 in use of the two tails of the test.

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The experiment included 20 rats receiving HES-A, 20 receiving HES-B, and 21 receiving HES-C. The interstitial space of the cremaster muscle became bright after the start of re-infusion. FITC-HES leaked out from the blood vessels within 1 min after the FITC-HES re-infusion (Fig. 1). The diameters of the arterioles and venules were 44 ± 2 μm and 47 ± 2 μm, respectively.

Figure 1

Figure 1

The rates of retention of FITC-HES in the arteriolar and venular blood vessels differed among groups (Figs. 2 and 3). Figure 2 is a graphic representation of the change of the retention of FITC-HES inside the V2 blood vessel after the FITC-HES infusion over a 120-min period. HES-A was found to have decreased steadily. The fluorescent intensity became one-third of that at point zero in 2 h. The fluorescent intensities of HES-B and HES-C did not decrease as much as that of HES-A. The retention rates of HES-A, -B, and -C in the V2 vessel at 120 min were 27% ± 7.2%, 65% ± 9.1%, and 86% ± 9.6%, respectively, of baseline values. The decrements of fluorescent intensity of HES-B (P = 0.028) and HES-C (P = 0.022) were significantly smaller than that of HES-A. The retention rates of HES-B and HES-C were significantly greater than HES-A after 40 min.

Figure 2

Figure 2

Figure 3

Figure 3

The retention rate of FITC-HES inside the A2 blood vessel after a 120-min shock period is shown in Figure 3. In the arteriole as well, the FITC-HES decreased gradually in all groups, although the degrees were different. HES-C decreased most remarkably in the arteriole, as well as in the vein (V2). The retention rates of HES-A, -B, and -C in the A2 vessel at 120 min were 27% ± 6.6%, 73% ± 10.2%, and 89% ± 8.7%, respectively, of baseline values. The retention rate of HES-B (P = 0.038) and HES-C (P = 0.037) were significantly larger than HES-A.

The detection of the FITC-HES outside the V2 venules was also examined (Fig. 4). The fluorescent intensity of HES-A outside the V2 venules increased the most rapidly. Apparently the increase of HES-C was the smallest, and the increase of HES-B was in between that of HES-A and of HES-C as shown in the Figure 4. However, no significant difference was found among any of the groups, as shown in the Figure 4.

Figure 4

Figure 4

The detection of FITC-HES outside the A2 arteries is shown in Figure 5. The fluorescent intensity of HES-A increased most rapidly and that of HES-C increased least rapidly. The values of HES-B were always between those of HES-A and HES-C. However, no significant difference was found among any of the groups, as shown in Figure 5.

Figure 5

Figure 5

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Each HES solution is characterized by concentration, MW, and DS, where DS is the percentage of hydroxyethyl residue in the glucose subunits (1). Hydroxyethylation is mandatory because the natural starches are unstable and rapidly hydrolyzed in the circulating amylase. The hydroxyethyl residues, especially when bound at the C2 carbon position of glucose, reduce the ability of the plasma-amylase to degrade the glucose polymers, hence increasing the intravascular half-life of the HES particle. A higher MW range and more extensive DS result in slower elimination.

In Europe, various types of HES are available, with MWs ranging between 70 and 450 kDa and DS between 0.4 and 0.7. In the United States, however, only HES with a MW of 450 kDa and a DS of 0.7 (hetastarch) has been approved, and in Canada HES with a MW of 270 kDa and a DS of 0.5 (pentastarch) is available. In Japan, HES with a MW of 70 kDa and a DS of 0.5 (hespander) is commercially available (12).

We produced our FITC-HES from the three compounds. To our knowledge, this study is the first in which synthesized FITC-HES was detected by an intravital microscope (10,13,14). With regard to the recent classification of HES, two of the three compounds in our newly synthesized FITC-HES were of medium MW. The remaining HES had a high MW. Although HES of low MW (hespander) is currently available for clinical use in Japan, we were only able to obtain the three medium MW and high MW FITC-HESs used in this study.

It seemed reasonable that FITC-HES-C stayed for a long time in the vascular space because the MW was high. What was not easily accounted for were the different properties of HES-A and HES-B, both starches of middle MW. In our study, HES-B was retained longer in the vascular space than HES-A. The MWs of HES-A and HES-B averaged 175 and 200 kDa, respectively. Their DS were 0.64 and 0.5, and their C2/C6 ratios were 0.5 and 0.6, respectively. Possible explanations for why HES-B was retained within the vessel space longer are the 2.5 kDa difference in MW (although the difference seems subtle) (1,3,15), the distribution of the MWs of the two HES particles (16), the difference in DS values (17), and the difference in the C2/C6 ratios (18). In addition, we hypothesized that medium-sized HES with low DS can divide rapidly, but at a rate that maintains a constant osmotic pressure so that the starch remains longer in the vessel space. The middle DS value led to increased metabolic degradation, which might have been counteracted by the increased C2/C6 ratio, preventing HES-B from decreasing too rapidly in the plasma. We can only say for certain that we found a difference in the half-lives of the two HES of middle MW in the vascular space.

Although the HESs of high MW have a beneficial long-term volume effect, it is reported that large HESs cause several side effects. Recent European studies attempting to solve the hemostatic derangement report on the development of a newly synthesized middle-sized HES with low DS and high C2/C6 ratio (19–22). There is also one report of a large HES with low DS and a high C2/C6 ratio (23). In our study, we observed that one of the two middle-sized HES compounds we used was retained in the vascular space statistically as long as a HES with high MW. We reconfirmed that HES with middle MW and higher DS were resistant to leakage from the vascular space.

In our study, the vessels in the cremaster muscle viewed with microscopy could have been compromised by the pressure of the polyethylene slide cover, which was necessary to keep the muscle specimen from drying out. In the presence of the compromised endothelial integrity, starch molecules could leak out from the vessels to the interstitial space significantly (6). The values of the intravascular decrease of HES illumination could have been different form those under physiological conditions. Further investigations are required to determine the values.

In conclusion, we studied the retention rates of three different FITC-HESs using intravital microscopy. The middle-sized HES with a MW of 175–225 kDa, a DS of 0.45–0.55, and a C2/C6 = 6:1 was found to stay longer in the blood vessels than the middle-sized HES with a MW of 150–200 kDa, a DS of 0.6–0.68, and a C2/C6 = 8:1. The former HES demonstrated a retention rate comparable to that of the HES of high MW. The middle-sized HES with low DS would be beneficial to reduce side effects. Further investigations are necessary to determine the properties of the ideal HES.

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The authors sincerely thank Masahiro Shibata, PhD, and Prof. Akira Kamiya, MD, PhD (Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo) for their technical advice. The authors also thank Yukifumi Kokuba, PhD, of the Ajinomoto Pharma Co., Ltd., Tokyo, Japan, for providing the HES that they used in the present study.

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