Effects of Preoperative Flavonoid Supplementation on Different Organ Functions in Rats

Effects of Preoperative Flavonoid Supplementation on Different Organ Functions in Rats

van Hoorn, Danny E C

ABSTRACT. Background: Previously it has been reported that preoperative feeding preserves heart function in rats after intestinal ischemia-reperfusion. To further improve postoperative organ function, bioactive nutrition compounds were selected in vitro against the xanthine oxidase radical cascade, an enzyme suggested to play a key role in the induction of single- or multiple-organ dysfunction. Methods: FIavonoids were selected in vitro for their capacity to (1) inhibit xanthine oxidase, (2) scavenge superoxide, and (3) scavenge peroxylradicals. The most bioactive flavonoids were added to the preoperative nutrition to study their effect on postintestinal ischemia-reperfusion organ function. Results: A combination of flavonoids selected on basis of effective flavonoid xanthine oxidase inhibition and superoxide scavenging resulted in increased superoxide scavenging. In vivo, the selected flavonoid mixture significantly lowered postischemic intestinal apoptosis and intestinal oxidative stress indicated by malondialdehyde concentration when compared with ischemia-reperfusion fasted and sham-fasted animals. Moreover, this flavonoid mixture significantly lowered plasma creatinine and urea concentration, both indicating a better postoperative kidney function. Furthermore, oxidative stress measured as this flavonoid mixture when compared with control significantly lowered plasma malondialdehyde concentration in fed rats. Conclusions: Coadministration of bioactive flavonoid mixture to preoperative nutrition, in contrast to fasting, attenuates ischemia-reperfusion injury by preserving kidney function in the rat and decreasing apoptosis in the intestine. (Journal of Parenteral and Enterai Nutrition 30:302308, 2006)

Fasting before surgery, the standard procedure in Western Europe and many other countries, alters the metabolic response to stress,1-5 leading to a catabolic state. There are several clinical and experimental studies suggesting that preoperative feeding or early postoperative enterai feeding reduces, among others, postoperative complications and lean body mass.6-14 Previously, it has been reported that preoperative nutrition in contrast to fasting preserved heart function.15

Although it has been reported that preoperative feeding can improve postoperative complications, such as insulin resistance, little is known about factors in feeding causing this observed effect.

Postischemically, increase of xanthine oxidase (XO) enzyme activity can be an important mediator of postoperative oxidative stress after reperfusion.16-20 Increased oxidative stress might lead to increased organ injury, which eventually can lead to single- or multiple-organ dysfunction (S/MOD).21,22 Recent literature also suggests that a group of naturally occurring compounds called flavonoids, abundant in fruits and plants, may be potent inhibitors of XO.23-27 Some of these compounds have been reported to inhibit inducible nitric oxide synthase (iNOS)28-30 and to be involved in the reduction of inflammation.31″33 Both iNOS and general inflammation have been reported to promote S (MOD). We hypothesized that supplementation of preoperative food with a selected flavonoid mixture inhibiting the XO cascade (Figure 1) further decreases ischemia reperfusion (IR)-induced S (MOD).

The combination of flavonoids with the highest in vitro capacity were tested for their in vivo effectiveness when added to preoperative nutrition in a rat model of intestinal IR, as previously described.34 Postoperative clinical organ function parameters such as plasma urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine were measured in both a preoperative fasted group, a preoperative fed group, and a group supplemented additionally with the selected flavonoids. Also, the lipid peroxidation production of malondialdehyde (MDA) as indicator of oxidative stress was measured in rat plasma and intestine.


In Vitro Research

XO inhibition. XO (X1875, Sigma Aldrich, Zwijndrecht, NL) activity was measured as described previously.24 In short, uric acid production is measured calorimetrically at 295 nm at pH 7.8. The flavonoids (Indofine Chemical Company Inc, NY) were tested up to a maximum concentration of 40 µM. Inhibition was expressed as IC^sub 50^. Allopurinol was used a standard inhibitor to compare against the activity of the flavonoids.

Superoxide scavenging assay. The assay is based on the nitrate method, with a slight modification in the XO activity (2.42 mil instead of 1.45 mU). In this assay, vitamin C is used a control superoxide scavenger.

The peroxyl radical scavenging method. The oxygen radical capacity assay (ORCA) is based on the work done by Cao et al35 laboratory. In short, the assay compares the protective effect of compounds on the decay velocity of the fluorescent protein β-phycoerythrin (Sigma Aldrich) with the water-soluble vitamin E analog, Trolox (8 µmol/L; Sigma Aldrich). The reaction mix contained 50 µl 50 mmol/L phosphate buffered saline (PBS) pH 7.4, 50 µl 66.8 nmol/L β-phycoerythrin, and 10 µL sample (either in water or ethanol). The reaction was started by addition of 100 µl 64 mmol/L 2.2′-azobis(2-amidinopropane; AAPH, Wako Chemicals, DE) at 28°C, which forms peroxyl radicals upon decay. This reaction was allowed to proceed to completion. Data are expressed in Trolox equivalents (TE) on a molar basis in which a compound with TE >1 is considered to be positive.

In Vivo Research

Ethical committee. The animal ethical committee of the Free University Medical Center (Amsterdam, NL) approved the animal experiments according to the Declaration of Helsinki.

Surgical details. Intestinal IR was applied as previously described.34 In short, the surgery was started by performing a laparotomy, followed by clamping the superior mesenteric artery for 60 minutes, followed by a reperfusion period of 180 minutes.

Male Wistar rats (250-350 g; Harlan, NL) were acclimatized for 7 days upon arrival, followed by a 7-day period of measuring body weight and food intake (semisynthetic feeding) twice a day (Table I). Five days before surgery, animals were divided into 4 groups.

The first group (sham-fasted, ç = 7) was allowed ad libitum access to food and drinking water until 13 hours before surgery. After 13 hours, the food was removed and water remained ad libitum. The shamfasted group received only a laparotomy and did not undergo ischemia. The second group (n = 10), referred to as the IR-fasted group, received the same preoperative procedure as the sham-fasted group and in addition underwent clamping of the superior mesenteric artery (SMA) for 60 minutes, resulting in IR injury of the intestine. The third group (n = 9), referred to as the IR-fed group, was allowed ad libitum access to food and drinking water until 2 hours before surgery and underwent the same clamping as experienced by the rats in the IR-fasted group. The fourth group (n = 9) was treated similarly as the third group (n = 9). In addition, the fourth group was supplemented with selected flavonoids, starting 5 days before surgery (Table II). The mixture was comprised of 4 g of quercetin, 3 g of luteolin, 3 g of apigenin, 5 g of epicatechin, and 10 g of green tea extract (Flachsmann, 085.0942, DE) added to 15 kg of standard feeding (Arie Blok Diervoeders). Assuming a food intake of 15 g per day estimates a flavonoid mixture intake of 25 mg. All rats were killed at the end of the 180-minute reperfusion period or the equivalent period in animals not subjected to IR injury (Table I), as described previously.34 For analyses of intestinal apoptosis, untreated ad libitum-fed animals (n = 4) were used as control because histology suggested shortened villi in the fasted groups, which impaired histologie quantification on apoptosis between fasted and fed groups.

Oxidative stress assessment. MDA concentration in plasma and intestine was measured as previously described by high performance liquid chromatography (HPLC).34

Apoptosis. Apoptosis was measured using the TUNEL assay.

Samples. Blood with 2% 0.275 M Na EDTA was centrifuged at 2600 X g at 4°C for 20 minutes, and plasma was used to determine urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate, creatinine, MDA. The organs were assessed for MDA concentration, as described previously.34

Statistics. ANOVA was used for statistical analysis of the data by using SPSS version 11.0. The data were tested for normality by the Kolmogorov-Smirnov and the Shapiro-Wilk tests. The data were also tested by Levene’s test of equality of error variance. The results are presented as means ± SEM unless indicated otherwise. As a post hoc test, the Student’s ß-test was used; p


In Vitro Experiments

First, the flavonoids (Figure 2) were selected on basis of their XO inhibition out of a pool of >30 flavonoids, as previously described.24 As can be seen in Table II, the 3 most active flavonoids found were quercetin, apigenin, and luteolin. second, a concentration range of flavonoids was tested for their superoxide scavenging capacity using XO as a radical producing system. Only those flavonoids that showed an IC^sub 50^ value lower than expected from direct XO inhibition were considered as positive. As can be seen in Table II, epicatechin and the green tea extract showed superoxide-scavenging properties that even exceeded the capacity of ascorbic acid. Third, as can be seen in Table II, all 4 pure compounds showed good peroxyl-scavenging properties and may therefore provide an additional protection against peroxyl radicals. Finally, whether the components could be combined was tested. Figure 3 shows that a flavonoid mixture of both the most effective XO inhibitors and superoxide scavengers provided an additive effect on decreasing the production of superoxide, suggesting components are still active when combined.

In Vivo Experiments

To test if our hypothesis implicating that preoperative feeding with selected in vitro XO cascade inhibiting flavonoids may decrease IR injury, 4 groups of rats were included (Table I). In short, group I was sham fasted and served as control. Group II was also fasted and additionally received IR and served as reference for post-IR injury when rats were fasted. The third and fourth groups were both allowed feed until the operation. The third group received a standard feeding and served as reference for post-IR injury when rats were preoperatively fed. The fourth group received an additional mixture of flavonoids selected for their in vitro XO cascade inhibition.

In order to establish whether intestinal ischemia did result in the induction of organ injury, several organinjury and organ-function parameters were measured, and the effects of preoperative feeding with addition of a selected flavonoid mixture on these parameters were assessed.

Plasma Parameters of Kidney Function

As reported earlier,34 the intestinal IR model used resulted in higher plasma concentration of urea and creatinine (Table III; p

Plasma Parameters of Liver Function and Lactic Acidosis

IR resulted in increased ALT and lactate (Table III; p

Intestinal Apoptosis and Oxidative Stress Assessment

Intestinal apoptosis of preoperatively fed rats was significantly higher than the intestines of control rats (p

Intestinal oxidative stress, as measured by tissue MDA levels, was significantly increased in preoperatively fasted group II. This negative effect of preoperative fasting was completely abolished in the flavonoidsupplemented group, which showed a significant decreased MDA concentration when compared with IR fasted animals (p

Systemic Parameters of Oxidative Stress

As can be seen in Figure 6, the flavonoid mixture significantly decreased total body oxidative stress expressed as plasma MDA concentration (p


During ischemia, the supply of oxygen and nutrients is severely diminished, especially in organs with a high-energy expenditure, which are rapidly forced to make energy (ATP) in a less efficient, anaerobic, manner.36″ This transition to anaerobic use of fuel initiates the XO cascade (Figure 1), starting with the increase of the ATP breakdown product, hypoxanthine (HX).36’39 This process is associated with an increase in lactate concentration and sodium influx in cells.40-44 A sustained period of ischemia may therefore lead to apoptosis or necrosis of 1 or more organs. To prevent organs from failure, the supply of oxygen has to be restored during a reperfusion period. Paradoxically, a reperfusion period can aggravate the effect of the ischemie period.19,23,24,45-47 This study focuses on the role of the XO cascade in this effect, according to reports on the importance of XO as an inducer of oxidative stress48 via conversion of xanthine dehydrogenase (XDH) to XO during intestinal IR, leading to increased oxidative stress by increased superoxide (Figure 1). This pathway in oxidative stress is emphasized by the increase of mRNA concentrations of both XO and XDH increase after ischemia.49 Also, XO can bind to a specific XO receptor present on endothelial cells,50 rendering it inaccessible to free radical scavengers such as superoxide dismutase. This increased production of superoxide may initiate a XO-induced radical cascade (Figure 2), leading to the production of many different reactive oxygen species (ROS). Therefore, the role for XO in IR induced damage is well acknowledged.18,19,26,47,49,51-58

Flavonoids were selected for their individual capacity to interfere at 3 levels with the initiation and prolongation of the XO induced radical cascade (Figure 1).1-5 As described in a previous study, flavonones, a subgroup in the flavonoid family, are effective xanthine inhibitors, being even superior in vitro to allopurinol.24 Because an increased XO activity results in an increased superoxide production (Figure 1), flavonoids were also selected for their superoxide scavenging capacity. A combination of the most effective XO inhibitors and superoxide scavengers (Figure 3) showed, in a concentration range that is achievable in plasma, an additional effect on superoxide scavenging.59-61 The most effective XO inhibitors and superoxide scavengers furthermore showed good peroxyl scavenging capacity and might therefore influence the XO radical producing systems in vivo at 3 multiple levels (Figure 2). We therefore selected these flavonoids for their possible effectiveness upon organ function improvement and biochemical parameters in vivo.

In the rat intestinal IR injury model, the plasma urea has frequently been reported to be increased during IR. This has been confirmed in the present study in which the IR fasted animals showed significantly increased plasma urea concentration compared with sham-fasted animals. Whether this increased urea is a result of reduced clearance, increased proteolysis, or increased production (arginase) remains to be investigated. Kidney function was significantly better in rats supplemented with flavonoids than in control fasted rats as indicated by lowered plasma creatinine and urea concentration (Figure 4A and 4B, respectively).

Addition of the flavonoid mixture protected the intestine from injury; this is seen by the significantly increased apoptosis level in preoperatively fed animals, whereas the flavonoid-fed animals showed apoptosis levels comparable to healthy control intestines. There are many factors influencing intestinal apoptosis levels; for instance, hampering gut blood flow or increased ROS and reactive nitrogen species (RNS). In this model, oxidative stress expressed as MDA concentration was more significantly lowered by addition of flavonoids to the preoperative feeding. This decrease in oxidative stress may indicate that at least part of the decreased apoptosis may be due to lowered oxidative stress.

The obtained data suggest that the flavonoid mixture used in this study has an additive protective effect over that seen with feeding alone. The flavonoid mixture even appears to correct a phenomenon of preoperative feeding that could be interpreted as negative, namely, the increased total body oxidative stress. This increase is evident from the plasma MDA concentration. The addition of the flavonoid mixture resulted in significantly lowered MDA concentration to a level that is similar to that in sham-treated animals.

In conclusion, these data strongly suggests that the addition of selected flavonoids to preoperatively fed animals provides an effective protection of kidney function and reduces intestinal apoptosis in a rat IR injury model, to a higher extent than seen in normal preoperatively fed animals. This implies that preoperative intake of nutrition additionally supplemented with selected XO cascade inhibitors is a more efficient manner to prepare a rat for intestinal IR injury than feeding alone. The question remains whether this rat data may have the same implications for humans undergoing operations.


Financial support for this study was provided by BTS grant BTS98020. We thank Nico van Munster and Rob Erik Blank for assistance on MDA measurement.


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Danny E. C. van Hoorn, PhD*; Robert J. Nijveldt, PhD[dagger]; Petra G. Boelens, MSc[dagger]; Zandrie Hofman*; Paul A. M. van Leeuwen, MSc, PhD[dagger]; and Klaske van Norren, PhD*

From *Numico Research, BMR, Wageningen, the Netherlands; and the [dagger]VU University Medical Center, Department of Surgery, Amsterdam, the Netherlands

Received for publication December 28, 2005.

Accepted for publication March 29, 2006.

Correspondence: Dr K. van Norren, Numico Research BV, PO Box 7005, 6704 PH Wageningen, The Netherlands. Electronic mail may be sent to Klaske.vanNorren@Numico-research.nl.

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