S Osowska

N Neveux

S Nakib

V Lasserre

L Cynober

C Moinard

Impairment of arginine metabolism in rats after massive intestinal resection : Effect of parenteral nutrition supplemented with citrulline versus arginine

Keywords: malnutrition, citrulline, arginine, short bowel syndrome, intestinal adaptation

Background: Arginine homeostasis is impaired in short bowel syndrome, but its supplementation in short bowel syndrome patients remains controversial. Recently, we demonstrated the benefits of citrulline supplementation by enteral route in resected rats. Since the first step in managing short bowel syndrome is to initiate total parenteral nutrition, we hypothesized that parenteral citrulline supplementation would be more appropriate in this situation than arginine supplementation. Material and methods: 24 rats were assigned to 4 groups. The Sham group underwent transection while the 3 other groups underwent resection (R) of 80% of the small intestine. All rats were then fed exclusively by total parenteral nutrition as follows: supplementation with citrulline (R+CIT), with arginine (R+ARG), or no supplementation (R). All the rats received isocaloric and isonitrogenous nutrition for 4 days.

Nitrogen balance was measured daily. At sacrifice, the blood was collected and the intestinal mucosa and Extensor Digitorum Longus muscle were removed for amino acid and protein analysis. Results: Citrulline and arginine increased mucosal protein content in the ileum (vs. Sham and R, p<0.05). However, only citrulline prevented Extensor Digitorum Longus atrophy (R+CIT: 130±3 vs. R: 100±6 and R+ARG: 110±2 mg, p<0.05). In addition, arginine worsened nitrogen balance (R+ARG: 104±46 vs. R: 249±69 mg/72 hrs, p<0.05). Conclusion: only citrulline was able to prevent muscle atrophy and it was achieved independently from any noticeable effect on the gut in particular because citrulline and arginine share the same effect on mucosal ileal protein content. These data suggest that citrulline should be considered as a potential supplement for total parenteral nutrition of short bowel syndrome patients.

Introduction

Arginine (ARG) presents numerous metabolic functions as a precursor of nitric oxide, agmatine, proline, polyamines and creatine [START_REF] Wu | Metabolic and therapeutic aspects of amino acids in clinical nutrition[END_REF]. Furthermore, ARG stimulates hormone secretion (i.e. insulin and growth hormone) [START_REF] Collier | Growth hormone responses to varying doses of oral arginine[END_REF]. Finally, it plays a key role in nitrogen homeostasis through the control of ureagenesis [START_REF] Cynober | Arginine metabolism in mammals[END_REF].

ARG homeostasis results from tight cooperation between the intestine and the kidneys, and citrulline (CIT) has a central role in this homeostatic process. At whole-body level, the intestine is the main site of CIT production because it is the only tissue that contains all the enzymes required to synthesize CIT from either ARG or glutamine (GLN; see schema) [START_REF] Curis | Almost all about citrulline in mammals[END_REF].

The kidneys (which express argininosuccinate synthase and argininosuccinate lyase) extract about 75% of plasma CIT, which represents 83% of the CIT released by the gut [START_REF] Wakabayashi | Metabolic and therapeutic aspects of amino acids in clinical nutrition[END_REF][START_REF] Dhanakoti | Renal arginine synthesis: studies in vitro and in vivo[END_REF][START_REF] Castillo | Plasma arginine and citrulline kinetics in adults given adequate and arginine-free diets[END_REF][START_REF] Castillo | Plasma arginine, citrulline, and ornithine kinetics in adults, with observations on nitric oxide synthesis[END_REF].

These metabolic considerations explain why short bowel syndrome (SBS) is characterized by profound perturbations of ARG metabolism [START_REF] Wakabayashi | Metabolic and therapeutic aspects of amino acids in clinical nutrition[END_REF] and why some authors [9;10] have suggested that ARG is in fact an essential amino acid in SBS. However, we recently demonstrated [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF] that CIT supplementation is more efficient than ARG in increasing ARG pools and restoring nitrogen balance after massive intestinal resection in rats. In our previous work, CIT was administered by enteral route. Since the liver and the intestine play a major role in ARG metabolism, it may be hypothesized that the route of administration (i.e. parenteral route, which bypasses the splanchnic area, versus the enteral one) may profoundly affect the metabolism of the administered CIT and ARG. The effect would probably be more pronounced on ARG than CIT, which is neither metabolized in the gut nor taken up or released by the liver [1;3]. The aim of this work was to evaluate the effects of CIT and ARG administered by parenteral route in a rat model of massive intestinal resection.

Materials and Methods

Chemicals: All chemicals were purchased from Sigma (St-Quentin-Fallavier, France).

CIT was a gift from Biocodex (Compiègne, France).

Animals: Male Wistar rats (n = 24, body weight 220-230 g) purchased from Charles River (L'Arbresle, France) were housed in metabolic cages for a 5-day acclimatization period before surgery, and were given free access to standard laboratory chow (17% protein, 3% fat, 59% carbohydrate, 21% water, vitamins and minerals) (A04, Usine d'Alimentation Rationnelle, Villemoisson-sur-Orge, France) and water.

Surgical procedures:

The animals were fasted for 12 h before surgery. They were anesthetized by isoflurane inhalation (3% isoflurane) using a regulated airflow apparatus (Minerve, Esternay, France). Enterectomy was performed on the rats by removing 80% of the small intestine, leaving 10% of the proximal jejunum and 10% of the distal ileum, as previously described [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF]. Six rats were sham-operated (transection). For both resected and transected animals, continuity was restored with end-to-end anastomosis using a single running silk 6-0 suture. Immediately after intestinal resection or transection, the catheter was fitted into the jugular vein. A silicone tube (Tygon, size 0.51 mm, Fisher Bioblock Scientific, Illkirch, France) was introduced into the vein and the remaining end of the catheter was tunneled subcutaneously to the back of the neck and attached to a spring coil-swivel mechanism (Harvard Apparatus, Les Ulis, France) allowing the rats to move freely within the cage [12;13].

Before the surgical procedure, all the rats received a painkiller (Temgesic ® , Schering-Plough, Levallois-Perret, France) by the subcutaneous route at a dose of 0.05 mg/kg body weight. Postoperative care: The rats were housed individually in metabolic cages. Parenteral nutrition was started immediately after surgery. Parenteral nutrition was given using a Harvard infusion pump (Pump 11, Harvard Apparatus, Les Ulis, France). During the first 24 hours of parenteral nutrition, the flow rate was increased progressively to reach a final rate of 300 ml/kg body weight/day, which corresponds to a nitrogen intake of 2 g/kg/day and 260 kcal/kg/day energy, covering nutritional requirements in rats [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF][START_REF] Osowska | Efficiency of a cysteine-taurine-threonine-serine supplemented parenteral nutrition in an experimental model of acute inflammation[END_REF][START_REF] Loi | Evaluation of a glycine-rich amino acid solution for parenteral nutrition in endotoxemic rats[END_REF]. The rats had free access to water.

Experimental groups:

The rats were randomly assigned to one of four groups:

• In the citrulline group (R+CIT, n = 6), the rats received standard parenteral nutrition (Vintène ® , Baxter, Maurepas, France) (Table 1) supplemented with CIT at a dose of 1 g/kg/day (5.71 mmol/kg/day).

• In the arginine group (R+ARG, n = 6), the rats received the standard parenteral nutrition supplemented with ARG at a dose of 0.994 g/kg/day, making it equimolar to the CIT group.

• In the control group (R, n = 6) and the sham group (Sham, n = 6), the rats received standard parenteral nutrition and were either resected (R) or transected (Sham).

In all groups, energy was supplied as carbohydrate (glucose 50 %) and long-chain triglycerides containing fat emulsion (Endolipid 20 %, B. Braun Medical, Boulogne, France). The nutritional regimen also provided electrolytes (Standard III, Aguettant, Lyon, France) and water. All the groups received isonitrogenous (2g N/kg/day, adjusted adding Vintene ® in R, sham and CIT groups) and isocaloric (260 kcal/kg/day) nutrition. Nitrogen and calorie intakes and amounts of CIT or ARG mimicked those used in clinical nutrition, which takes into account the fact that metabolic rate is ten-fold higher in rats than in humans [11;14;15]. The rats were weighed and urine was collected daily. Total parenteral nutrition was administered for 4 days. Parenteral nutrition was stopped 2 h before sacrifice by decapitation.

Tissue removal

Blood was sampled into heparinized tubes, which were rapidly centrifuged.

Jejunum and ileum: The intestine was resected from the ligament of Treitz to the ileocecal junction. The mucosa was washed with NaCl (0.9 %), everted and scraped off, then weighed, rapidly frozen in liquid nitrogen and stored at -80 ° C until analysis.

Muscles:

The extensor digitorum longus (EDL) was rapidly removed , weighed, then frozen in liquid nitrogen and stored at -80°C until analysis.

Parameters under study and analytical methods

Amino acid (AA) concentrations in plasma and tissues: Plasma was deproteinized with a 30% (w/v) sulfosalicylic acid solution and the supernatants were stored at -80°C until analysis.

Tissues were ground and deproteinized with 10% trichloroacetic acid (TCA) containing 0.5 mM EDTA, and the supernatants were stored at -80°C until analysis. AAs were measured by ion exchange chromatography [START_REF] Neveux | Measurement of amino acid concentration in biological fluids and tissues using ion-exchange chromatography[END_REF] using an amino acid analyzer (AminoTac, JLC-500/V, Jeol, Tokyo, Japan). The results of our participation in the European Quality Control Scheme (ERNDIM, Maastricht, The Netherlands) indicate the accuracy of our AA determinations.

Polyamines: Dansyl derivatives of intestinal polyamines were assayed according to the method of Seiler and Knodgen [START_REF] Seiler | High-performance liquid chromatographic procedure for the simultaneous determination of the natural polyamines and their monoacetyl derivatives[END_REF], with some modifications. Intestine samples were homogenized in a phosphate-buffer (50 mM KH 2 PO 4 /Na 2 HPO 4 , 300 µl of buffer per mg of tissue).

Homogenates were centrifuged for 5 min at 3000 g. Supernatants were diluted 15 times in phosphate buffer and stored at -80°C until analysis. Samples were incubated for 2 hours at 60°C in the dark with 0.5 ml of dansyl chloride (5 mg/ml in acetone). The solution was then evaporated under nitrogen and extraction was performed using 8 ml of 30% methanol (v/v).

The organic phase was collected and evaporated, and the dansyl derivatives were resuspended in 0.1 ml of the initial mobile phase and 20 microliters were injected into the chromatograph.

Dansylputrescine, dansylspermidine and dansylspermine were separated on a C8 column (length: 15 cm, particle diameter: 5 µm) using the 5500 Vista Varian pump system (Varian, Palo Alto, USA). After fluorimetric detection, polyamine concentrations were estimated by the internal standard method (standard: hexanediamine). Results are expressed as nmol per gram of tissue.

Nitrogen was quantified by chemiluminescence [START_REF] Grimble | Measurement of elemental nitrogen by chemiluminescence [letter; comment[END_REF] using an Antek 9000 apparatus (Antek, Houston, TX), and nitrogen balance was calculated as the difference between nitrogen intake and urinary nitrogen output.

Statistical analysis

Data are expressed as means + SEM. Comparisons between sets of data were made using oneway analysis of variance (ANOVA) followed by the Duncan test. PCSM software was used (Deltasoft, Grenoble, France). Differences at p < 0.05 were considered significant. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Results

Body weight loss (Figure 1) (i.e. the difference between body weight at the beginning of TPN administration and the end of the experiment) did not differ significantly in the four studied groups.

Tissue weight (Table 2). Gut resection led to muscle atrophy (R vs. Sham, p<0.05). However, EDL weight was restored by CIT supplementation (R+CIT vs. R and R+ARG, p<0.05). ARG and CIT supplementation increased mucosal ileum weight (R+CIT and R+ARG vs. Sham, p<0.05) whereas only the CIT-treated group had higher mucosal jejunum weight than sham (vs. Sham, p<0.05). 3). There were no significant changes in protein content in EDL muscle. Intestinal resection led to a significant increase in mucosal protein content in the jejunum (R vs. sham, p<0.05) without a significant additional effect of ARG or CIT supplementation. The mucosal protein content of the ileum, which was not modified by resection, was significantly increased in both the ARG-and CIT-supplemented groups (R+ARG and R+CIT vs. Sham and R, p<0.05). Amino acid concentrations. CIT was the only amino acid whose profile was influenced by resection or by the different diets (Table 4A).

Tissue protein contents (Table

Plasma. Intestinal resection led to a decrease in CIT levels compared with the sham group (R vs. Sham, p < 0.05). CIT administration led to an increase in plasma CIT concentrations (R+CIT vs. Sham, R, R+ARG, p < 0.05). In the CIT group, the increase in EDL weight was positively correlated with plasma CIT concentration (r = 0.73, p<0.0001).

Muscles. Intestinal resection led to a decrease in muscle CIT levels (R vs. Sham, p<0.05). Only CIT supplementation led to a significant increase in muscle CIT levels (R+CIT vs. Sham, R, R+ARG, p<0.05).

Intestinal mucosa. After intestinal resection, there was a decrease in CIT content in the mucosal jejunum (R vs. Sham, p<0.05). While both CIT and ARG led to an increased CIT content in the mucosal jejunum, only CIT supplementation was able to completely restore mucosal jejunum CIT content (R+CIT vs. R and R+ARG, p<0.05).

There were no treatment effects on ARG levels (Table 4B) nor on other AAs (data not shown).

Polyamine contents in intestinal mucosa (Table 5). Intestinal resection was associated with a significant increase in spermidine (R vs. Sham, p<0.05). This higher level of spermidine content was also found in the mucosal jejunum from the CIT-supplemented group (R+CIT vs. Sham: p<0.05) but not in the ARG-supplemented group (R+ARG vs. Sham: ns). There was no difference in spermine or putrescine contents between the groups studied.

Nitrogen balance (Figure 2). Cumulative nitrogen balance was significantly lower in the three resected groups compared with the Sham group (Sham vs. R, R+ARG and R+CIT, p<0.05), especially when the diet was supplemented with ARG (R+ARG vs. R, p<0.05).

DISCUSSION

The intestine is an important organ for the maintenance of ARG homeostasis and therefore for whole-body nitrogen homeostasis. The pioneering study by Crenn et al [START_REF] Crenn | Postabsorptive plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal failure in humans[END_REF] together with earlier experimental data [9;10] suggest that ARG administration could become critical in SBS.

In fact, the metabolic specificities of ARG and its related AAs (see introduction) together with our recent research [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF] show that it is CIT administration rather than ARG which could be a better strategy in an SBS setting.

As expected, there was a two-fold increase in plasma CIT concentrations after parenteral CIT administration. This result is in agreement with our previous work using enteral infusion [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF].

However, CIT supplementation did not affect ARG concentration in either the plasma or in the tissues. This is a surprising result since we previously observed that the same amount of CIT (i.e. 1 g/kg/d) given by enteral route is able to generate a large amount of ARG in resected animals [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF]. The main factor determining ARG synthesis by the kidney is plasma CIT concentration [START_REF] Dhanakoti | Renal arginine synthesis: studies in vitro and in vivo[END_REF]. Since renal function, assessed on the basis of urinary creatinine, was not altered in our model (data not shown), it is likely that ARG synthesis was not compromised.

Data from the literature [5;7;8] indicate that ARG catabolism is the main regulator of ARG homeostasis. Using a similar model of enterectomy and labelled isotopes, Dejong et al [START_REF] Dejong | Renal arginine metabolism in fasted rats with subacute short bowel syndrome[END_REF] demonstrated, that net renal CIT uptake and ARG release were both decreased by 50% due to intestinal resection. However, despite decreased net renal CIT consumption and renal ARG production in enterectomized rats, neither the whole-body rate of ARG appearance nor arterial ARG were modified, prompting the same group [START_REF] Welters | Effects of parenteral arginine supplementation on the intestinal adaptive response after massive small bowel resection in the rat[END_REF] to conclude that intestinal resection does not have important effects on whole-body ARG production. Therefore, our data clearly show that ARG administration failed to increase ARG pools. The dosage used cannot be incriminated since the same dose (0.994 g/kg/day) given by enteral route in the same SBS rat model was associated with a large increase in plasma and muscle arginine content [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF]. This suggests that ARG is intensively metabolized in response to intestinal resection and that ARG metabolism is clearly affected by route of administration (Sham + standard enteral nutrition: ARG: 110 ± 12 µmol/L (from [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF]) and Sham + standard parenteral nutrition: ARG: 246 ± 24 µmol/L). This may reflects the importance of splanchnic first-pass in ARG metabolism.

However, the time schedule of the present study was different from our earlier one [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF] and so comparison of data requires caution. The few data available on parenteral supplementation in SBS rats [21;22] actually did not use the same route of administration (i.e. ARG was administered subcutaneously, which does not reflect an intravenous administration). Another explanation is that the rats were studied earlier than in our previous study [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF] making the rats more catabolic with higher ARG turnover. Finally, in certain pathological situations, the liver appears to be able to extract CIT [23;24] by diverting this amino acid from kidney capture. The results we obtained using the enteral route [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF] and the parenteral route, show that CIT reaching the liver by the arterial artery has a different behavior from CIT coming from the portal vein. This point deserves further studies. Furthermore, studies using labeled amino acids and arterio-venous difference measurements are required to define ARG use at whole-body and organ levels, following resection.

Our results suggest that ARG and CIT increase mucosal weight and mucosal protein content. We also observed that these effects could differ according to the intestinal section (jejunum or ileum). This difference may be related to differences in the adaptation process that

are not yet understood. These data are promising results which suggest that CIT and ARG could affect gut protein content. It would be interesting to extend our investigation to specific markers of intestinal adaptation (i.e. bowel circumference, bowel weight, mucosa DNA content, villus height and crypt depth) and to determine the mechanism involved in this action.

The effect of ARG on intestinal adaptation remains controversial [9;10;20;22;25;26]. At the molecular level, the synthesis of intracellular polyamines is a critical event for the onset of mucosal hyperplasia. Hence, these molecules play an important role in the maintenance of intestinal mucosa [START_REF] Moinard | Polyamines: metabolism and implications in human diseases[END_REF]. This is well illustrated in SBS, since a significant increase in the polyamine content of the remaining intestine was reported in the literature [20;28]. However, in our study, only spermidine was increased in the jejunum (R and R+CIT vs Sham, p<0.05).

This discrepancy with the literature may be because polyamine content usually increases in the first hours following resection (less than 24 hours) [29;30]. In the present study, polyamine assays were performed 96 hours after resection, and the absence of the significantly increased polyamine content after this period is consistent with the studies cited above.

Alternatively, ARG, when given parenterally, may be used preferentially by other tissues. As expected, resected animals exhibited a decreased nitrogen balance. While CIT had no effect on nitrogen balance, ARG clearly appeared to have a deleterious effect. This could be explained as follows: as arginase activity is very high in the liver [START_REF] Morris | Arginine: beyond protein[END_REF], most of the ARG that reaches the liver is converted into urea. This fact was well illustrated by de Bandt et al.'s study [START_REF] De Bandt | Metabolism of ornithine, alpha-ketoglutarate and arginine in isolated perfused rat liver[END_REF] in a model of isolated perfused liver. These authors demonstrated that ARG was extensively taken up by the liver and rapidly released as ORN and urea, which supports ARGinduced acceleration of amino acid catabolism and activation of the urea cycle. However, CIT, which is not taken up by the liver [START_REF] Drotman | Citrulline metabolism in the perfused rat liver[END_REF], limits the activation of ureagenesis, making it a candidate therapy for preserving nitrogen balance. However, this study using parenteral administration did not reproduce the beneficial effect of CIT on nitrogen balance observed when CIT is administered by enteral nutrition [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF]. As for the ARG levels, this discrepancy may be explained by the difference in the duration of the treatment (3 days in this work vs. 10 days previously), because the beneficial effect of CIT was observed only after the 4 th day of treatment.

Finally, EDL weight was increased in the CIT-supplemented group compared to both the control and ARG-supplemented groups. Interestingly, this increase was positively correlated with plasma CIT concentrations (r = 0.73, p<0.0001). This unexpected funding could be related to the ability of CIT to stimulate muscle protein synthesis [START_REF] Moinard | Citrulline: a new player in the control of nitrogen homeostasis[END_REF]. We recently showed [START_REF] Osowska | Citrulline modulates muscle protein metabolism in old malnourished rats[END_REF] that CIT administration to malnourished old rats stimulates muscle protein synthesis, which could explain the trend in increased muscle protein content observed in the present study. In addition, our recent research [START_REF] Moinard | Citrulline directly modulates muscle protein synthesis: evidence from in vitro studies[END_REF], using isolated incubating muscles suggests that CIT is able to directly affect protein synthesis. However, to the best of our knowledge, our work is pioneering and there is no data available in the literature that might help us to explain this effect. Alternatively, a modification of water content might be involved. Unfortunately, this variable was not measured in this study. However, we previously measured muscle water content in a number of studies in several physiopathological conditions (aging, enteral or parenteral nutrition, stress, malnutrition, SBS, etc.) and it was never modified by treatment.

The present work does not enable us to draw definitive conclusion: further work to assess body composition (using DEXA or D 2 O methods), whole body and tissues protein synthesis would help to determine the precise influence of CIT on lean body mass in various tissues and organs.

In conclusion, parenteral CIT supplementation restores the plasma CIT pools that are reduced after intestinal resection. However, our results are different from those obtained after enteral administration. This suggests that the route of administration (i.e. enteral vs. parenteral) has profoundly different effects on ARG metabolism. CIT shares with ARG the positive effect on intestinal adaptation in terms of intestinal mucosa weight and protein content. However, only CIT also has a positive effect on muscle weight and importantly, ARG administration worsens nitrogen balance, whereas CIT does not. These data suggest that CIT could be potentially considered as a supplement in parenteral nutrition for the patients with SBS in order to preserve nutritional state and promote intestinal adaptation. Together with our previous work [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF], these results open the way to clinical trials in patients suffering from SBS. Four groups of rats were studied: Sham (transected rats fed standard parenteral nutrition), R (resected rats fed standard parenteral nutrition), R+ARG (resected rats fed an arginine-supplemented formula), R+CIT (resected rats fed a citrulline-supplemented formula) Results are expressed as means ± SEM. ANOVA + Duncan's test. Values in a row with different superscript letters are statistically different at p<0.05. Four groups of rats were studied: Sham (transected rats fed standard parenteral nutrition), R (resected rats fed standard parenteral nutrition), R+ARG (resected rats fed an arginine-supplemented formula), R+CIT (resected rats fed a citrulline-supplemented formula).

Results are expressed as means ± SEM. ANOVA + Duncan's test (GLNase, Glutaminase; OAT, ornithine aminotransferase; OCT, ornithine carbamoyltransferase; ARGase, arginase; ARG, arginine; CIT, citrulline; ORN, ornithine; GLN, glutamine; GLU, glutamate; ASS+ASL, argininosuccinate synthetase + lyase).

  One of the authors (C.M.) is authorized by the French government (No. 75522) to use this experimental model of surgery. Animal care complied with French regulations for the protection of animals used for experimental and other scientific purposes (D 2001-486) and with European Community regulations (Official Journal of the European Community L538 12:18:1986).

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 11 Jan 2008 as manuscript CS20070451 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070451

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 11 Jan 2008 as manuscript CS20070451 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070451

Fig 1 .

 1 Fig 1. Cumulative body weight loss over the duration of total parenteral nutrition (g).

Fig 2 .

 2 Fig 2. Nitrogen balance over the duration of total parenteral nutrition (mg/72h). Four groups of rats were studied: Sham (transected rats fed standard parenteral nutrition), R (resected rats fed standard parenteral nutrition), R+ARG (resected rats fed an arginine-supplemented formula), R+CIT (resected rats fed a citrulline-supplemented formula). Results are expressed as the difference between daily nitrogen intake and daily urinary nitrogen excretion. Results are expressed as means ± SEM. ANOVA + Duncan's test. Values in a row with different superscript letters are statistically different at p<0.05.

Table 2 . Muscle and intestine mucosa weight.

 2

		Sham	R	R+ARG	R+CIT
	EDL (mg)	120±6 a,c	100±6 b	110±2 a,b	130±3 c
	Jejunum (mg/cm)	36±6 a	48±6 a,b	52±3 a,b	65±9 b
	Ileum (mg/cm)	26±2 a	44±5 a,b	54±3 b	52±9 b
	Four groups of rats were studied: Sham (transected rats fed standard parenteral nutrition), R
	(resected rats fed standard parenteral nutrition), R+ARG (resected rats fed an arginine-
	supplemented formula), R+CIT (resected rats fed a citrulline-supplemented formula)
	Results are expressed as means ± SEM.
	ANOVA + Duncan's test. Values in a row with different superscript letters are statistically
	different at p<0.05.

Table 3 . Protein content in the intestinal mucosa and EDL muscle.

 3

		Sham	R	R+ARG	R+CIT
	EDL (mg)	11.9±1.9	10.1±1.4	11.9±0.9	13.6±0.8
	Jejunum (mg/g)	46±3 a	57±7 b	72±4 b	73±6 b
	Ileum (mg/g)	51±3 a	53±7 a	71±5 b	72±3

b

Four groups of rats were studied: Sham (transected rats fed standard parenteral nutrition), R (resected rats fed standard parenteral nutrition), R+ARG (resected rats fed an argininesupplemented formula), R+CIT (resected rats fed a citrulline-supplemented formula) Results are expressed as means ± SEM. ANOVA + Duncan's test. Values in a row with different superscript letters are statistically different at p<0.05.

Table 4 . Citrulline (A) and arginine (B) concentrations in plasma and tissues.

 4

	A-Citrulline	Sham	R	R+ARG	R+CIT
	Plasma (µmol/L)	80±6 a	46±2 b	41±5 c	114±7 d
	EDL (nmol/g)	167±16 a	79±7 b	100±16 c	502±27 d
	Jejunum (nmol/g)	145±25 a	59±10 b	63±17 c	143±33 a
	Ileum (nmol/g)	141±35	53±13	86±21	156±37
	B-Arginine
	Plasma (µmol/L)	246±24	228±31	244±25	288±19
	EDL (nmol/g)	1200±163	1071±204	951±122	1131±184
	Jejunum (nmol/g)	304±63	444±80	467±66	200±61
	Ileum (nmol/g)	200±58	401±113	256±62	190±31
	Four groups of rats were studied: Sham (transected rats fed standard parenteral nutrition), R
	(resected rats fed standard parenteral nutrition), R+ARG (resected rats fed an arginine-
	supplemented formula), R+CIT (resected rats fed a citrulline-supplemented formula)
	Results are expressed as means ± SEM.
	ANOVA + Duncan's test. Values in a row with superscript letters are statistically different
	at p<0.05.

Table 5 . Polyamine content in the intestinal mucosa.

 5

	Jejunum	Sham	R	R+ARG	R+CIT
	putrescine	55±14	89±11	70±12	84±14
	spermidine	697±62 a	1004±46 b,c	821±57 a,c	987±100 b,c
	spermine	401±20	520±25	392±33	444±67
	Ileum	Sham	R	R+ARG	R+CIT
	putrescine	44±4	68±29	128±23	86±28
	spermidine	879±87	951±20	1048±80	909±131
	spermine	614±27	526±73	479±29	499±78

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 11 Jan 2008 as manuscript CS20070451 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070451 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 11 Jan 2008 as manuscript CS20070451 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070451 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S. Osowska was the recipient of a fellowship from Laboratoires Biocodex. This research was partly supported by a grant from Laboratoires Biocodex and partly by a grant from the French Ministry of Research and Technology (4-year contract EA 2498).


Dr Patrick Linsel-Nitschke
email: patrick.linsel-nitschke@uk-sh.de

MD Anika Götz

MSc Anja Medack

MSc Inke R König

PhD Petra Bruse

Wolfgang Lieb

MD Björn Mayer

MD Klaus Stark

PhD Christian Hengstenberg

MD Marcus Fischer

MD Andrea Baessler

MD Andreas Ziegler

PhD Heribert Schunkert

MD Jeanette Erdmann

Genetic variation in the arachidonate 5-lipoxygenase-activating protein (ALOX5AP) is associated with myocardial infarction in the German population

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Despite numerous advances in treatment, coronary artery disease (CAD) and its major complication myocardial infarction (MI) still represent the leading causes of mortality in the Western world [START_REF] Keys | Seven countries: A multivariate Analysis of Death and Coronary Heart Disease[END_REF]. Both environmental and genetic factors contribute to the pathogenesis of CAD and MI. Genetic epidemiological approaches including genome-wide linkage and association studies identified several chromosomal regions and polymorphisms related to MI and/or CAD [START_REF] Wang | Molecular genetics of coronary artery disease[END_REF][START_REF] Samani | Genomewide association analysis of coronary artery disease[END_REF][START_REF] Mayer | Genetics and heritability of coronary artery disease and myocardial infarction[END_REF]. However, independent validations as well as mechanistic explanation of the underlying functional basis for many of these findings are still under investigation.

In two recent studies, Helgadottir and coworkers reported a strong genetic association between the ALOX5AP gene and risk of MI and stroke in the Icelandic population [START_REF] Helgadottir | The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke[END_REF][START_REF] Helgadottir | A variant of the gene encoding leukotriene A4 hydrolase confers ethnicity-specific risk of myocardial infarction[END_REF].

ALOX5AP encodes for arachidonate 5-lipoxygenase-activating protein, which plays a key role in the biosynthesis of pro-inflammatory leukotriene B mediators providing a potential link between inflammation and cardiovascular disease. In a genome-wide scan of 296 multiplex Icelandic families including 713 individuals with MI suggestive linkage lead to the identification of the ALOX5AP gene as a strong candidate gene for MI. Within the ALOX5AP gene the authors identified a four SNP haplotype (haplotype A) conferring a two-fold risk for MI. In functional studies using isolated neutrophils from carriers of haplotype A, an increased production of the pro-inflammatory leukotriene B was demonstrated [START_REF] Helgadottir | The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke[END_REF]. However, the association of haplotypes A and B of the ALOX5AP gene with leukotriene B production was not confirmed in a recent study [START_REF] Maznyczka | Leukotriene B4 production in healthy subjects carrying variants of the arachidonate 5-lipoxygenaseactivating protein gene associated with a risk of myocardial infarction[END_REF].

In a subsequent report, the same authors who first reported the association of haplotype A with MI in the Icelandic population were unable to confirm this association in a cohort of British patients. However, the authors identified another 4 SNP haplotype of the ALOX5AP gene, termed haplotype B, which conferred significant risk of both MI and stroke [START_REF] Helgadottir | Association between the gene encoding 5-lipoxygenase-activating protein and stroke replicated in a Scottish population[END_REF].

Similarly, a recent angiography based study from Italy has detected an increased risk of angiographically proven CAD for carriers of haplotype B but not for carriers of haplotype A [START_REF] Girelli | ALOX5AP gene variants and risk of coronary artery disease: an angiographybased study[END_REF]. In contrast to the aforementioned European studies no association of either haplotype A or B of the ALOX5AP gene with MI was detected in further studies from the US[10] and Japan [START_REF] Kajimoto | Validation of the association between the gene encoding 5-lipoxygenase-activating protein and myocardial infarction in a Japanese population[END_REF].

The LTA4H gene encodes for leukotriene A 4 hydrolase, a protein in the same biochemical pathway as FLAP, the gene product of ALOX5AP. A subsequent study reported a moderately increased risk of MI for carriers of a haplotype in the LTA4H gene (haplotype K) [START_REF] Helgadottir | A variant of the gene encoding leukotriene A4 hydrolase confers ethnicity-specific risk of myocardial infarction[END_REF] in the Icelandic population. In a similar fashion as previously demonstrated for ALOX5AP, the authors were again able to show an increased production of pro-inflammatory leukotriene B associated with haplotype K of the LTA4H gene [START_REF] Helgadottir | A variant of the gene encoding leukotriene A4 hydrolase confers ethnicity-specific risk of myocardial infarction[END_REF]. In the same report, the authors replicated the association of haplotype K with MI and stroke in pooled populations from three North-American cities comprising 1,591 MI cases with European ancestry and 197 MI cases with African American ancestry. The increased chance conferred by haplotype K was markedly higher in African Americans and for subjects with MI and concomitant cerebrovascular disease [START_REF] Helgadottir | A variant of the gene encoding leukotriene A4 hydrolase confers ethnicity-specific risk of myocardial infarction[END_REF].

Since the published data on the association of ALOX5AP with CAD is conflicting and no further studies on the association of genetic variation in LTA4H with MI have been reported so far, we decided to investigate the association of genetic variation in both genes with MI in the German MI Family Study.

Methods

Study population

MI families were ascertained through patients at 17 cardiac rehabilitation centers distributed throughout Germany. All patients had suffered from a MI before 60 years of age [START_REF] Fischer | Distinct heritable patterns of angiographic coronary artery disease in families with myocardial infarction[END_REF]. If at least one sibling presented with MI or severe CAD (defined as percutaneous coronary intervention or coronary artery bypass grafting) before 70 years of age, the nuclear family (patient, available parents, and all affected and unaffected siblings) was contacted and invited to participate in the study. All study participants answered to a standardized questionnaire about medical history, presence of coronary risk factors, clinical events, medication, anthropometric data, and socioeconomic background. This information was validated by retrospective analyses of medical records. Additionally, all patients underwent a medical examination during a visit scheduled at their primary care physician's office. For this study, we examined 1,211 unrelated MI patients. 1,015 healthy married-in spouses served as a control group.

The study protocol was approved by the local institutional review-committee, and all subjects gave written informed consent.

Genotyping

SNPs comprising haplotype A (consisting of the common allele of rs17222814 (G), the common allele of rs10507391 (T), the common allele of rs4769874 (G), and the rare allele of rs9551963 (A)) and haplotype B (rs17216473, rs10507391, rs9315050, rs17222842) in the ALOX5AP gene and one SNP in the LTA4H gene (rs2660899) were genotyped using 5´exonuclease activity (TaqMan) on a ABI Prism 7900HT (Applied Biosystems, Darmstadt, S t a g e 2 ( a ) P O S T -P R I N T Germany). TaqMan genotyping assays with probes labelled with the fluorophores FAM and VIC, respectively, were purchased from Applied Biosystems. Genotyping was performed on 384-well plates prepared with the GENESIS Freedom pipetting robot from TECAN (Crailsheim, Germany). The Universal PCR Master Mix from Applied Biosystems was used in a 5 µl total reaction volume with 10 ng DNA per reaction. Allelic discrimination was measured automatically on the ABI Prism 7900HT (Applied Biosystems) using the Sequence Detection Systems 2.1 software (autocaller confidence level 95%).

Ten percent of all genotypes were repeated in independent PCR reactions to check for consistency and to ensure intra-and inter-plate genotype quality control. No genotyping discrepancies were detected between the repeated samples.

Statistical analysis

We performed power analyses based on the odds ratios (OR) provided from the literature for haplotypes A and B [START_REF] Helgadottir | The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke[END_REF]. For this, we applied the two-sided exact test of Fisher at a global testlevel of 5%.

Deviations of genotype distribution from and compatibility with Hardy-Weinberg equilibrium (HWE) were analyzed using a Monte Carlo χ 2 goodness-of-fit test (treshold of significance every p > 0.05, HWEχ 2 ) and the exact uniformly most powerful equivalence test with δ 2 = 2 and δ 1 = 2/3, respectively, for all patient groups and all control groups for all markers separately [13].

Primary aim of the study was to show the association between ALOX5AP and LTA4H genes and MI. Therefore we examined for the ALOX5AP gene the two haplotypes A and B that are sufficient to cover genetic variation in this gene according to Helgadottir and coworkers [START_REF] Helgadottir | The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke[END_REF].

Given that a previous study has demonstrated that SNP rs2660899 in the LTA4H gene provides the best single surrogate marker of haplotype K, which is associated with the greatest risk of MI [START_REF] Helgadottir | A variant of the gene encoding leukotriene A4 hydrolase confers ethnicity-specific risk of myocardial infarction[END_REF], we have only genotyped rs2660899 for the LTA4H gene. p-values of the haplotype analyses were adjusted for multiple testing according to Bonferoni-Holm [START_REF] Holm | A simple sequentially rejective multiple test procedure[END_REF] and were considered as significant if they were smaller than 5%.

Analyses of haplotypes A and B were performed using the EM-algorithm. Here, score statistics based on a χ 2 distribution with one degree of freedom and corresponding p-values were calculated as proposed by [START_REF] Schaid | Score tests for association between traits and haplotypes when linkage phase is ambiguous[END_REF] [15]. We determined Monte-Carlo pvalues from 1,000,000 replications.

In addition, for every SNP of haplotype A and B separately as well as for rs2660899, differences in genotype frequencies between MI cases and controls were tested using the 2- The authors had full access to the data and take responsibility for its integrity. All authors have read and agreed to the manuscript as written.

Results

Baseline characteristics of the study population are shown in Table 1.

For the power analysis, we assumed a haplotype frequency of 15.8%. for haplotype A and an odds ratio of 1.8, as reported in the initial study [START_REF] Helgadottir | The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke[END_REF]. This resulted in sufficient power to detect differences (99.9%), even a more moderate effect of 1.4 is detectable with our study (power = 85.6%). The power to detect an odds ratio of 1.95, as reported in the initial study for haplotype B with a frequency of 7.5%, was 99.7%.

To evaluate genotyped SNPs for HWE, we checked for all patient and control groups for all markers separately if there are deviations of genotype distribution with a Monte Carlo χ 2 goodness-of-fit test (Table2). No deviation from HWE was detected this way (every p > 0.05, HWEχ 2 ). Second, we analyzed the compatibility with HWE using the exact uniformly most powerful equivalence test with δ 2 = 2 and δ 1 = 2/3, respectively (HWE aequi). For SNPs rs4769874 and rs9315050 we detected slightly more heterozygotes in the cases than expected.

However, such findings are not unusual in cases in regions linked to a disease. The frequency of haplotype A in our study population was comparable with those reported in the literature [START_REF] Helgadottir | The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke[END_REF][START_REF] Helgadottir | Association between the gene encoding 5-lipoxygenase-activating protein and stroke replicated in a Scottish population[END_REF][START_REF] Girelli | ALOX5AP gene variants and risk of coronary artery disease: an angiographybased study[END_REF]. However, we detected no association between MI and haplotype A (Monte-Carlo p = 0.2807, showed a significant association with MI (each Monte-Carlo p > 0.05). In contrast, haplotype B (consisting of the rare allele of rs17216473 (A), the rare allele of rs10507391 (A), the common allele of rs9315050 (A), and the common allele of rs17222842 (G)) was significantly associated with MI (Monte-Carlo p < 1x10 -5 , Table 3b). Also, the global association score test of these four SNPs showed a significant association (Monte-Carlo p = 2x10 -5 ). Further, the last SNP of the haplotype rs17222842 probably is the most important SNP, because the single SNP analysis also gives a hint for an association (p = 0.0175; OR per 1 rare allele [95%CI]: 0.78 [0.63,0.96]) and the haplotype AAAA is also associated with MI (Monte-Carlo p = 0.0112). Contrary to the haplotype AAAG that is associated with increased risk of MI (haplotype score = 5.0), the haplotype AAAA shows a protective effect (haplotype score = -2.5).

In addition, we performed subgroup analyses for haplotype A and B of the ALOX5AP gene and rs2660899 of the LTA4H gene (Table 4). Whereas we did not detect a significant association of either haplotype A or rs2660899 in any subgroup, the effect of haplotype B was found in every subgroup investigated.

Discussion

In analogy to two previous reports from Great Britain [START_REF] Helgadottir | Association between the gene encoding 5-lipoxygenase-activating protein and stroke replicated in a Scottish population[END_REF] and Italy [START_REF] Girelli | ALOX5AP gene variants and risk of coronary artery disease: an angiographybased study[END_REF], we detected an increased risk of MI for carriers of haplotype B of the ALOX5AP gene in our German study whereas no association of haplotype A with MI risk was observed. Haplotype frequency, odds ratio and direction of effect for haplotype B in our population proved to be very similar to the previous reports from Britain [START_REF] Helgadottir | Association between the gene encoding 5-lipoxygenase-activating protein and stroke replicated in a Scottish population[END_REF] and Italy [START_REF] Girelli | ALOX5AP gene variants and risk of coronary artery disease: an angiographybased study[END_REF] but different to the North American study in which no difference in frequency of haplotype B was detected between MI cases and controls [10]. The fact that the association of haplotype B with risk of CAD has now been replicated three times in European populations but proved negative in the North American study might hint at differences in genetic structure, namely differences in SNP linkage disequilibrium and haplotype structure between European and North American populations.

For the LTA4H gene our study did not find any association of SNP rs2660899 representing haplotype K with risk of MI in the German population. In the original report the risk associated with haplotype K was particularly pronounced in individuals suffering from MI with concomitant cerebrovascular disease and in African American individuals. However, a subgroup analysis for individuals with MI and concomitant stroke in our German study did not yield any additional information. The lack of association between rs2660899 representing haplotype K with MI in the German MI study consisting entirely of Caucasians might again In summary, our study was able to confirm the association between a common haplotype of ALOX5AP and CAD in the European population. There is now increasing evidence from several studies for a role of ALOX5AP in the pathogenesis of CAD, particularly in subjects of European descent.

Table legends

Table 1: Baseline characteristics of the study population stratified by affection status.

Displayed are means ± standard deviations and frequencies in percent.

Table 2: Overview of single-SNP analyses in the ALOX5AP and LTA4H genes .

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Mar 2008 as manuscript CS20070468 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070468 sided Cochrane-Armitage trend test; ORs and corresponding 95% confidence intervals (CI) were calculated. For the haplotypes A and B and rs2660899, we furthermore performed additional subgroup analyses investigating patients who were either suffering from MI before 45 or 50 years of age or those patients with MI who had a positive history of stroke or other cerebrovascular disease (history of transient ischemic attack or known supraaortic stenosis), because previous studies had indicated that the association of polymorphisms in both genes with risk of cardiovascular disease is markedly increased in individuals with early-onset MI or in individuals affected by both MI and concomitant cerebrovascular disease.

Frequencies

  and minor allele frequencies (MAF) of the single SNPs comprising haplotype A and B of the ALOX5AP gene and rs2660899 of the LTA4H gene are displayed in Table 2. In addition, p-values of Cochrane-Armitage trend test; and corresponding ORs plus 95% CIs are shown. No association of rs2660899 with MI was detected.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Mar 2008 as manuscript CS20070468 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070468 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Mar 2008 as manuscript CS20070468 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070468 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society --be due to population specific effects. Further studies in populations of different ethnic origins might be needed in order to better understand the association of genetic variation in LTA4H with MI and stroke.

Table 4 :

 4 Genotype abbreviation: 0= homozygous for common allele, 1=heterozygous, 2=homozygous for rare allele; HWE χ 2 = pvalue of Monte Carlo χ 2 goodness-of-fit test for deviation from HWE; HWE aequi = confidence intervals for heterozygotes for compatibility with HWE using the exact uniformly most powerful equivalence test with δ 2 = 2 and δ 1 = 2/3, respectively. P-trend: unadjusted, 2-sided p-values of Cochrane-Armitage trend test; OR indicates odds ratio; 95% CI indicates 95% confidence intervals of OR. Table 3a: Association of haplotype A in ALOX5AP with MI. Displayed are global and specific haplotype scores. Analyses were performed in 1,211 MI Cases and 1,015 controls. Haplotype A is shaded in grey. Table 3b: Association of haplotype B in ALOX5AP with MI. Displayed are global and specific haplotype scores. Analyses were performed in 1,211 MI Cases and 1,015 controls. Haplotype B is shaded in grey. Subgroup analyses of haplotypes A and B in ALOX5AP and rs2660899 in LTA4H. Displayed are global and specific haplotype scores for the haplotypes. For rs2660899 MAF, 2-sided p-values of Cochrane-Armitage trend test; ORs and 95% CI are presented. Each subgroup was analysed separately versus the 1,015 controls. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Mar 2008 as manuscript CS20070468 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070468

S t a g e 2

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S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Mar 2008 as manuscript CS20070468 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070468 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  Table 3a) despite sufficient power. Accordingly, neither the other haplotypes of the four SNPs comprising haplotype A, nor the global association score test

Table 1 MI

 1

		Cases	Controls
	N	1211	1015
	Men (%)	782 (64.6)	377 (37.1)
	Age, years (%)	58.2 ± 8.6 56.8 ± 9.9
	Body mass index, kg/m 2	27.5 ± 3.7 26.7 ± 4.2
	Systolic blood pressure, mm Hg 138.0 ± 19.7 134.0 ± 17.5
	Diastolic blood pressure, mm Hg 82.4 ± 10.3 82.1 ± 9.8
	Total cholesterol, mg/dl	227.7 ± 46.0 238.3 ± 42.3
	HDL cholesterol, mg/dl	50.6 ± 13.3 60.5 ± 15.3
	LDL cholesterol, mg/dl	151.1 ± 42.5 147.5 ± 34.5
	Smoking status (%)	822 (67.9)	509 (50.1)
	Diabetes (%)	197 (16.3)	56 (5.5)
	Antihypertensive medication (%) 1066 (88.0)	382 (7.6)
	Lipid lowering medication (%) 801 (66.1)	94 (9.3)

Table 2 .

 2

		RS17222814	RS10507391		RS4769874	RS9551963
	Position	30197553	30210096		30224441	30230547
	Major/Minor Allele	G/A		T/A			G/A	C/A
	Gene	ALOX5AP -Hap A	ALOX5AP -Hap A, Hap B	ALOX5AP -Hap A	ALOX5AP -Hap A
		Cases	Controls	Cases	Controls	Cases	Controls	Cases	Controls
	0	903	819	488	463	971	862	302	245
	1	216	153	474	389	101	76	526	455
	2	10	9	120	100	1	3	290	190
	freq of rare allele	10.5	8.7	33.0	30.9	4.8	4.4	49.5	46.9
	HWE χ 2	0.5232	0.5751	0.7849	0.1741	0.5064	0.4154	0.0538	0.4164
	HWE aequi	[196,220]	[147,159]	[384,562]	[329,473]	[97,99]	[76,78]	[436,682]	[349,539]
	p Trend	0.05675	0.1656		0.5027	0.1118
	OR [95% CI]*	1.22	[0.99,1.51]	1.10	[0.96,1.25]	1.11	[0.82,1.49]	1.11	[0.98,1.25]
	OR [95% CI]**	1.50	[0.99,2.27]	1.20	[0.93,1.56]	1.23	[0.68,2.22]	1.22	[0.95,1.56]

Table 2 (

 2 continued)

		RS17216473	RS9315050	RS17222842	RS2660899
	Position	30201965	30234045	30238117	94932888
	Major/Minor Allele	G/A		A/G		C/T		G/T
	Gene	ALOX5AP -Hap B	ALOX5AP -Hap B	ALOX5AP -Hap B	LTA4H
		Cases	Controls	Cases	Controls	Cases	Controls	Cases	Controls
	0	820	740	966	834	889	735	770	622
	1	270	228	162	125	170	188	322	278
	2	29	12	3	8	10	10	43	34
	freq of rare allele	14.7	12.9	7.4	7.3	8.9	11.1	18.0	18.5
	HWE χ 2	0.2787	0.2592	0.1901	0.1583	0.5730	0.6235	0.2283	0.7381
	HWE aequi	[248,298]	[200,230]	[148,158]	[125,133]	[164,178]	[170,192]	[290,364]	[244,306]
	p Trend	0.09297	0.8659	0.0175	0.6534
	OR [95% CI]*	1.16	[0.98,1.39]	1.02	[0.81,1.29]	0.78	[0.63,0.96]	0.96	[0.83,1.13]
	OR [95% CI]**	1.35	[0.95,1.93]	1.04	[0.65,1.66]	0.6	[0.40,0.92]	0.93	[0.68,1.27]

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Mar 2008 as manuscript CS20070468 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070468 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society --13

Table 3a .

 3a

		SNPs	rs17222814 (G/A), rs10507391 (T/A), rs4769874 (G/A) , rs9551963 (C/A)
	Global	Nominal p				0.2067
	Score	Simulated p				0.2058
		Haplotype	GTGC	GTGA	GAGA	ATGA	GAGC	GAAC
	Specific Score	Haplotype frequency Haplotype Score Nominal p	42.5 -1.9 0.0579	15.4 -1.1 0.2804	23.3 1.2 0.2257	9.5 1.9 0.0532	4.5 0.5 0.62244	4.2 0.4 0.6906
		Simulated p	0.0577	0.2807	0.2249	0.0533	0.62312	0.6907
	Table 3b.
		SNPs	rs17216473 (G/A), rs10507391 (T/A), rs9315050 (A/G) , rs17222842 (G/A)
	Global	Nominal p				1 x 10 -5
	Score	Simulated p				1 x 10 -5
		Haplotype	GTAG	GAAG	AAAA	AAAG	GAGG	GTGG	GTAA
	Specific Score	Haplotype frequency Haplotype Score Nominal p	63.8 -1.5 0.1376	13.3 0.6 0.5733	8.1 -2.5 0.0113	5.3 5.0 < 0.0001	4.9 0.0 0.9822	2.2 0.1 0.9281	1.3 -0.1 0.8826
		Simulated p	0.1377	0.5737	0.0112	< 0.0001	0.9822	0.9285	0.8833

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Table 4 .

 4 OR per 1 rare allele [95% CI] 0.96 [0.83,1.13] 0.98 [0.77,1.26] 1.01 [0.80,1.28] 1.01 [0.83,1.23] rs2660899 OR per 2 rare alleles [95% CI] 0.93 [0.68,1.27] 0.97 [0.59,1.58] 1.03 [0.64,1.65] 1.02 [0.69,1.51]

	Early onset MI

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Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

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Acknowledgements:

This study was supported by the Deutsche Forschungsgemeinschaft (Schu672/9-1, Schu672/10-1, Schu672/12-1 and Schu672/14-1), the Federal Ministry of Research (Dr. Schunkert, KBF-FKZ 01GB9403), the National Genome Network (01GS0418 to Drs Schunkert, Erdmann, and Hengstenberg; 01GR0466 to Dr. Ziegler), The Ernst-and Berta-Grimmke-Stiftung (Drs. Hengstenberg and Schunkert), the Wilhelm-Vaillant-Stiftung (Drs. Hengstenberg and Schunkert), the Deutsche Stiftung für Herzforschung (Drs. Hengstenberg and Schunkert), and the EU sponsored project Cardiogenics (LSHM-CT-2006-037593). S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Mar 2008 as manuscript CS20070468 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070468 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society --


Dan Berndtson

Joel Olsson

Robert G Hahn
email: r.hahn@telia.com

Professor Robert G Hahn Head

Hypovolaemia after glucose-insulin infusions in volunteers

Keywords: glucose, insulin, osmolality, hypovolaemia, hypoglycaemia

High-dose intravenous infusion of glucose 5% promotes rebound hypoglycaemia and hypovolaemia in healthy volunteers. To study whether such effects occur in response to glucose-insulin, 12 healthy firemen (mean age 39 years) received 3 infusions over 1-2 hours that contained 20 ml/kg of glucose 2.5%, 5 ml/kg of glucose 10% with rapid-acting insulin 0.05 U/kg, and 4 ml/kg of glucose 50% with 1 U/kg of insulin. The plasma glucose concentration and plasma dilution were compared at 5-10 min intervals during 4 hours. Regardless of the amount of administered fluid and whether insulin was given, the plasma glucose concentration decreased to hypoglycaemic levels within 30 min after the infusion ended. Plasma dilution closely mirrored plasma glucose and became negative by about 5%, which indicates a reduction of the plasma volume. These alterations were only partially restored during the follow-up period. A linear relationship between plasma glucose and plasma dilution was most apparent when the infused glucose had been dissolved in only a small amount of fluid. For the strongest glucose-insulin solution, this linear relationship had a correlation coefficient of 0.77 (n=386, P< 0.0001). The findings indicate that a redistribution of water due to the osmotic strength of the glucose is the chief mechanism accounting for the hypovolaemia. It is concluded that infusions of glucose 2.5%, 10% and 50% with and without insulin in well-trained men were consistently followed by longstanding hypoglycaemia and also by hypovolaemia, which averaged 5%. These results emphasise the relationship between metabolism and fluid balance.

INTRODUCTION

Glucose-insulin-infusions have a role in anaesthesia and intensive care, although the precise indications are debated. Glucose-insulin is used to combat cardiac insufficiency in cardiovascular surgery [START_REF] Svedjeholm | Rationale for metabolic support with amino acids and glucose-insulin-potassium (GIK) in cardiac surgery[END_REF][START_REF] Szabó | Effects of high-dose glucose-insulin-potassium on myocardial metabolism after coronary surgery in patients with type II diabetes[END_REF] and might be given before general surgery begins as a mean of reducing the insulin resistance, surgical stress response and the incidence of nausea [START_REF] Ljungqvist | Glucose infusion instead of preoperative fasting reduces postoperative insulin resistance[END_REF][START_REF] Hausel | Randomized clinical trial of the effects of oral preoperative carbohydrates on postoperative nausea and vomiting after laparoscopic cholecystectomy[END_REF]. Insulin given together with 10% glucose infusions resulted in a reduction of mortality in the intensive care unit in the "van den Berghe study" [START_REF] Van Den Berghe | Intensive insulin therapy in critically ill patients[END_REF] and beneficial effects have been reported after acute myocardial infarction [START_REF] Diaz | Metabolic modulation of acute myocardial infarction[END_REF][START_REF] Marano | Effects of infusion of glucoseinsulin-potassium on myocardial function[END_REF]. In animals, glucose-insulin makes the myocardium utilise glucose instead of fatty acids as energy substrate, which might protect from permanent injury in ischaemic situations [START_REF] Zhu | Glucose-insulinpotassium preserves systolic and diastolic function in ischaemia and reperfusion in pigs[END_REF][START_REF] Korvald | Myocardial substrate metabolism influences left ventricular energetics in vivo[END_REF].

Glucose has an osmotic potential that affects the distribution of body water. Simple calculations suggest that each mmol of glucose attracts 3.6 ml of water, which is translocated from the extracellular to the intracellular fluid space along with the uptake of glucose by the cells, with or without the aid of exogenous insulin. The effects of glucose and glucose-insulin administration on the distribution of water have been poorly understood in the past. However, infusion of plain glucose 5% in volunteers was followed by mild hypovolaemia and hypoglycaemia 30-45 min after the infusion ended [START_REF] Sjöstrand | Volume kinetics of glucose solutions given by intravenous infusion[END_REF]. These effects were less apparent in those with a low glucose clearance, such as diabetic [START_REF] Sjöstrand | Intravenous hydration with a glucose 2.5% solution in type 2 diabetes[END_REF] and surgical patients [START_REF] Sjöstrand | Volume kinetics of 2.5% glucose solution during laparoscopic cholecystectomy[END_REF], but may possibly be aggravated by exogenous insulin.

The aim of the present study was to evaluate the severity and time-course of hypovolaemia developing in association with glucose-insulin therapy. On three occasions, volunteers were given an infusion with glucose 2.5% and electrolytes, a "mild" glucoseinsulin infusion and, finally, a "strong" glucose-insulin infusion. The first solution is frequently given during anaesthesia and surgery [START_REF] Sjöstrand | Intravenous hydration with a glucose 2.5% solution in type 2 diabetes[END_REF][START_REF] Sjöstrand | Volume kinetics of 2.5% glucose solution during laparoscopic cholecystectomy[END_REF], the second (glucose 10%) postoperatively [START_REF] Van Den Berghe | Intensive insulin therapy in critically ill patients[END_REF] while the third (glucose 50%) is often used during intensive care. The amounts of insulin used chosen were inspired by the practices of thoracic anaesthesia clinics in Sweden. The volunteers were closely supervised by our team and followed with both laboratory and bedside testing of the glucose level to allow mild but not severe hypoglycaemia to develop.

METHODS

After approval of the protocol by the Ethics Committee in Umeå, Sweden, 12 healthy male firemen aged between 26 and 50 (median 39) and with a body weight of 83 kg (72-100) agreed to participate in 3 laboratory experiments conducted at the intensive care unit (ICU) at a university hospital with an interval of at least one month. A 13th volunteer abandoned the study after the first experiment and was excluded from the analysis.

Experimental infusions

The volunteers received 3 infusions:

1. Only glucose; 20 ml/kg/h of iso-osmotic 2.5% glucose with electrolytes (Na 70, Cl 45 and acetate 25 mmol/l; Rehydrex, Pharmacia, Uppsala, Sweden) was infused over 1 hr. No insulin was given.

"

Mild" glucose-insulin infusion; 5 ml/kg/h of glucose 10% was given over 1 hr and rapid-acting insulin 0.1 U/kg/hr over 30 min (Actrapid, Novo Nordisk, Copenhagen, Denmark).

"Strong" glucose-insulin infusion; 2 ml/kg/h of glucose 50% over 2 hrs and

Actrapid insulin 1 U/kg/hr during 1 hr.

Due to the lag time before insulin exerts its effect, the insulin infusions were started 10 min before the glucose.

The three experimental infusions contained different amounts of fluid, glucose and insulin, and also a period of glucose infusion after insulin had been discontinued. Details are compared in Table 1.

All volunteers arrived at the ICU in the fasting state at 7 AM and received a light standard breakfast consisting of 200 ml of water or coffee and one sandwich. They were placed comfortably in a bed and, after instrumental preparation, they were left for haemodynamic equilibration over a period of 30 min. In the "only glucose" experiment, the infusions were given via a peripheral venous cannula, while the insulin-containing fluids were infused via central venous catheter placed in the axillary or subclavian vein via a cubital vein. Blood sampling was performed via an arterial catheter. A discard volume of 3 ml was drawn before each blood collection. This blood was then returned and the cannula flushed with 3 ml of acetated Ringer´s solution to prevent clotting.

Measurements

Blood was collected for measuring the blood haemoglobin (Hb) and plasma glucose concentrations, the haematocrit (Hct), mean corpuscular volume (MCV) and erythrocyte count (RBC) every 5 min up to 1.5 hrs, and every 10 min up to 4 hrs of each experiment.

All indices of the red cells were reported as the mean of duplicate samples, while the baseline was taken as the mean of triplicate samples.

The serum sodium, potassium and insulin concentrations were measured every 30 min. However, insulin was not measured during the "only glucose" series. In the "strong" glucose-insulin experiments, lack of adequate dilution of the samples before analysis made all the ones collected during the infusion show the maximum value for the kit, but all samples obtained from 1.5 hr and onward were within the range of detection.

The blood analyses were conducted by the certified standard routine chemistry laboratory at Umeå University Hospital.

The arterial blood pressure and the heart rate were measured via the arterial line every 5 min and displayed on a GE Solar 8000 M (General Electric, Milwaukie, Ore., USA). Voiding was allowed whenever needed, but the subject then remained in the recumbent position.

The volunteers were always attended by a registered nurse and by at least one member of the study team. Blood glucose was monitored bedside every 5 min by using a system which requires one drop of blood only for each analysis (HemoCue, Ängelholm, Sweden). Any sign of sweating and discomfort, which was checked continuously, as well as blood glucose levels of 2 mmol/l or lower, as detected by HemoCue, was treated with one or several intravenous "rescue" injections of glucose 30%. This was done in 19 experiments. Data on plasma glucose and the dilution obtained during and for 15 min after emergency injections were excluded from analysis. The research team had telephone contact with the volunteer at 24 hrs after each experiment. The volume of the "rescue" injections averaged 50 ml and the "flush" volume was 66 ml. The volume of sampled blood amounted to 300-350 ml.

Calculations

Glucose. The kinetics of the "only glucose" infusion was analysed to characterise how effectively the volunteers handle an exogenous glucose load. The pharmacokinetics was analysed using a one-compartment turnover model. The plasma concentration (C) at any time (t) is increased by an exogenous infusion rate (R i ex ) and the endogenous production of glucose (k i en ) which result in the glucose concentrations C ex and C en , respectively.

Hence, C = C ex + C en . The contribution of R i ex to the measured C was assessed from the following differential equation [START_REF] Sjöstrand | Volume kinetics of glucose solutions given by intravenous infusion[END_REF][START_REF] Sjöstrand | Intravenous hydration with a glucose 2.5% solution in type 2 diabetes[END_REF][START_REF] Sjöstrand | Volume kinetics of 2.5% glucose solution during laparoscopic cholecystectomy[END_REF]:

! dCex dt = Ri ex Vd " CL Vd * Cex(t) (Eqn. 1)
in which V d is the volume of distribution and CL the clearance of glucose. The glucose concentration resulting from endogenous production of glucose (C en ), which is responsible for the baseline concentration in these fasting volunteers, was simplified as:

! Cen = Ri en CL (Eqn. 2)
The model parameters (V d , CL and k i en ) were calculated from Eqn. 2 and the analytical solution Eqn.1 simultaneously on a PC using Matlab version 6.5 (Math Works Inc., Natick, MA, USA), operating with a non-linear least-squares regression routine based on a modified Gauss-Newton method was used [START_REF] Sjöstrand | Intravenous hydration with a glucose 2.5% solution in type 2 diabetes[END_REF]. Since glucose enters the cells by active transport, a decreasing amount of glucose in V d corresponds, in the absence of glucosuria, to the uptake of glucose into the body cells.

The half-life of the exogenous glucose was obtained as the product of V Plasma dilution = 0.5 Hbo /Hb(t) -1

1 ! Hcto + RBCo /RBC(t) -1 1! Hcto " # $ % & ' (Eqn. 3)
Before use, this expression was corrected for "iatrogenic" dilution resulting from the sampling (7 ml per sample) and also by the ratio MCV o /MCV(t) to account for changes in erythrocyte cell size [START_REF] Ewaldsson | Kinetics and extravascular retention of acetated Ringer´s solution during isoflurane and propofol anesthesia for thyroid surgery[END_REF].

Statistics

The results were presented as the mean and standard deviation (SD). The relationship between parameters was analysed by linear regression analysis. Significant changes from baseline were studied by repeated-measures ANOVA. P< 0.05 was considered statistically significant.

RESULTS

Plasma glucose and plasma dilution

In the "only glucose" experiments, the plasma glucose concentration decreased quite rapidly after the infusion was finished and remained below baseline (by P< 0.0001) from 30 min later and onward (Fig. 1, upper left). The half-life of the exogenous glucose averaged 7 min (Table 2).

Plasma dilution was also significantly below baseline (by P< 0.01; indicating haemoconcentration) from 30 min after the infusion ended (Fig. 1, upper right).

The "mild" glucose-insulin infusion resulted in a gradual reduction of the plasma glucose and the plasma dilution (Fig. 1, middle). Rescue injections of glucose 30% needed to be given to 7 volunteers after the infusion ended, despite the fact that insulin was stopped 30 min before the infusion of glucose 10%.

The "strong" glucose-insulin solution increased plasma glucose significantly only during the first hour of the 120-min infusion, and it dropped to below baseline within 30 min after it was stopped (Fig. 1, lower left). The plasma dilution could both increase and decrease during the infusion, but it decreased to significantly below baseline from 30 min later and up to the end of the study (P< 0.01 Fig 1, lower right; Table 3).

A linear relationship between individual changes in plasma glucose and plasma dilution was most apparent when a small volume of fluid accompanied the glucose infusion. Hence, this relationship was statistically significant during infusion of the "strong" glucose-insulin solution, between 0 and 30 min after, as well as >30 min after, these infusions ended (Fig. 2). The pooled data from all periods of time during infusion of the "strong" glucose-insulin solution further illustrated the consistent linear relationship between these parameters (Fig. 3).

The estimates of CL over time indicated that the CL was twice as high during the glucose-insulin infusions as compared to "glucose only" (Fig. 4, left).

Haemodynamics

With "glucose only", the diastolic arterial pressure increased gradually from baseline 66 Other findings include that nearly all of the fluid infused with the glucose was excreted during the experiments, which was hardly expected when considering the longstanding state of mild hypovolaemia. Haemodynamic responses consisted mainly in a modest increase of the heart rate and a decrease in arterial pressure after the glucoseinsulin infusions were ended. Reduction of serum potassium was a small issue, although glucose-insulin is usually provided together with potassium to prevent hypokalaemia from developing. Here, potassium was not added since it would increase the risk of pain on infusion of the strong glucose solutions.

The fact that haemoconcentration developed which corresponds to a reduction of the plasma volume by up to 15% (average 5%) can be explained by the osmotic strength of the glucose molecule. When administered intravenously, this molecule attracts water that is either recruited from the intracellular fluid space or else is provided with the infusion.

The fluid stimulates a diuretic response but the same fluid should also, by virtue of osmosis, accompany the glucose into the cells. The end-result of these events is a diuretic response and a matched load of glucose and water entering the cells. Hypovolaemia develops as the diuresis competes with the osmotic forces about the same fluid volume [START_REF] Sjöstrand | The osmotic link between hypoglycemia and hypovolemia[END_REF].

The glucose-insulin protocols used in this study are far from liberal. The Department of Thoracic Anaesthesia in Lund, Sweden, gives 50 E of insulin in 500 ml glucose 30% at a rate of 1.2 ml/kg/h. Our "mild" composition provided 30% more glucose and less than half as much insulin per hour but, to assure volunteer safety, the glucose infusion alone was continued for twice as long as the insulin infusion. The composition of the "strong" glucose-insulin infusion was derived from the Department of Thoracic Anaesthesia in Umeå, Sweden. Here, too, the glucose administration was continued for twice as long as the insulin infusion to limit the risk of hypoglycaemia. In both cases, the precautions proved to be insufficient. Several bolus injections of glucose 30% had to be given and the volunteers were often at the limit of experiencing blunt hypoglycaemia. However, the monitoring of the volunteers was elaborate. They were kept at the ICU of a university hospital and received attention similar to that given to cardiothoracic surgical patients. One drop of blood was also taken every 5 min for a bedside check-up of blood glucose, and the volunteers were asked to read a book and to report every experience of lost attention, blurred vision or nausea to the research team.

Our volunteers were well trained physically and had a glucose clearance (CL) of 0.8 litres/min when no insulin was provided, which is fairly high. Previous studies using the same one-compartment model yielded CL of 0.72 and 0.54 litres/min [START_REF] Sjöstrand | Volume kinetics of glucose solutions given by intravenous infusion[END_REF] and 0.6 litres/min [START_REF] Sjöstrand | Validation of volume kinetic analysis of glucose 2.5% solution given by intravenous infusion[END_REF] in healthy volunteers, which corresponded to half-lives for exogenous glucose of between 11 and 16 min. CL in patients with diabetes averaged 0.37 litres/min [START_REF] Sjöstrand | Intravenous hydration with a glucose 2.5% solution in type 2 diabetes[END_REF] and those undergoing laparoscopic cholecystectomy only 0.21 litres/min [START_REF] Sjöstrand | Volume kinetics of 2.5% glucose solution during laparoscopic cholecystectomy[END_REF]. Hence, while the two groups of patients had a half-life of glucose that was slightly longer than that for insulin, the opposite has been the case for all series of healthy volunteers in which rebound hypoglycaemia was also more apparent.

CL for the glucose-insulin experiments could not be calculated by conventional methods due to the many periods of hypoglycaemia. However, one purpose of conducting The longer half-life for insulin than for glucose might provide a hint about the risk of "rebound hypoglycaemia" when exogenous glucose administration is halted. The shorter half-life for glucose can, in turn, be understood from the logarithmic relationship between CL and the insulin concentration (Fig. 4, right) which maintains a rapid glucose elimination despite decreasing insulin concentrations.

The "rebound" phenomenon has been reported for infants whose mothers were given glucose infusions before delivery [START_REF] Philipson | Effects of maternal glucose infusion on fetal acid-base status in human pregnancy[END_REF][START_REF] Kenepp | Neonatal hazards of maternal hydration with 5% dextrose before caesarean section[END_REF] and is common in response to rapid infusion of glucose 5% in volunteers [START_REF] Sjöstrand | Volume kinetics of glucose solutions given by intravenous infusion[END_REF] as well as during intravenous glucose tolerance tests, which use glucose solutions with a strength of between glucose 20% and 50% [START_REF] Muniyappa | Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage[END_REF].

In the present study, rebound hypoglycaemia became apparent after infusing of glucose solution of very low strength, only 2.5%. A check-up of plasma glucose seems to be warranted 30 min after infusing a fixed combination of glucose and insulin, which is often done in the clinic to create a "safe" match between the two compounds.

The prolongation of the half-life for glucose associated with "insulin resistance" would make seriously ill and trauma patients less susceptible to rebound hypoglycaemia if glucose alone is infused [START_REF] Sjöstrand | The osmotic link between hypoglycemia and hypovolemia[END_REF]. Such resistance would cause a rightward shift of the relationship shown in the right panel of Figure 4. At very low and high CLs, this curve is also expected to flatten out [START_REF] Muniyappa | Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage[END_REF]. On the other hand, if insulin is also given the dose is likely to be raised to cope with any insulin resistance, as evidenced by the schemes used at the thoracic anaesthesia departments mentioned above. In those cases, it seems likely that rebound hypoglycaemia can occur despite insulin resistance.

Many views encouraged by the present results merit further study. Insulin promotes intravascular fluid loss in diabetes patients [START_REF] Gundersen | Intravenous insulin causing loss of intravascular water and albumin and increased adrenergic nervous activity in diabetics[END_REF] but we would like to know whether rebound hypoglycaemia is associated with hypovolaemia in other clinical conditions. This is important in patient groups that are more sensitive to hypovolaemia than others, such as trauma patients, septic, burns and those with chronic ischaemic heart disease. The reduction of plasma glucose created in patients treated for diabetic ketoacidosis might follow the same basic relationship as that shown in Fig. 3, which could then be used as a reference for rehydrating the ECF volume.

It is difficult to find a good alternative explanation to the hypovolaemia found in the present study other than rapid uptake of glucose by the cells, sustained by prolonged insulin action, which translocates water from the ECF to the intracellular fluid space by infusions would then represent a fluid deficit that is evenly distributed throughout the ECF volume, meaning that the deficit would amount to about 800 ml if the ECF volume equals 20% of the body weight. As 1 mmol of glucose attracts 3.6 ml of water, 800 ml corresponds well to the amount of fluid that can be expected to be translocated into the cells along with the infused glucose in the "fluid only" experiment.

The dehydration can probably be prevented by infusing additional sodium along with the glucose. A previously published sodium-fluid redistribution equation suggests that 200 mmol of sodium, or 160 ml of 7.5% saline, would offer sufficient prevention [START_REF] Drobin | Kinetics of isotonic and hypertonic plasma volume expanders[END_REF]. A benefit with sodium treatment is the relatively long duration of its plasma volume support, although blunt hypoglycaemia must still be carefully watched for. Another way to prevent the osmosis-induced hypovolaemia would be to provide fluid 30 min after the glucose load -infusing it together with the glucose will only result in a prompt diuretic response, as seen in our "glucose only" series of experiments.

The bolus injections of glucose 30% that were given to combat clinical hypoglycaemia resulted in major elevations of plasma glucose (15-25 mmol/l) and plasma dilution (10-15%), but they lasted only 10-15 min. Those data were not included in the figures or calculations because plasma dilution could lag one sample behind plasma glucose. This can be explained by the arterial line used for sampling while the blood needed to pass the capillaries before water could be recruited from the intracellular fluid space. However, these acute changes indicated that an increase in plasma glucose of 30 mmol/l corresponded to a plasma dilution of 20%, which further illustrates the osmotic strength of glucose.

In conclusion, the present results show that hypoglycaemia is associated with hypovolaemia which both developed 30 min after intravenous infusion of glucose with and without insulin in well-trained firemen. High glucose clearance despite falling insulin levels and the osmotic strength of glucose are proposed to be the key mechanisms explaining these findings.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  d and the natural logarithm of 2 (0.693) divided by CL. The CL during each 5-min or 10-min period was estimated for all 36 experiments by inserting the values of V d and R i en obtained during the "glucose only" experiments into Eqn. 1. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical SocietyThe serum half-life of insulin during the post-infusion period was obtained from the coefficient in a simple linear regression analysis of the relationship between the logarithm-transformed serum insulin concentration and time after the infusions ended.Plasma dilution. The dilution of arterial plasma was calculated based on the blood haematology obtained at baseline (time 0) and at any later time (t). The principle is to convert haemodilution into plasma dilution based on the average of the Hb and RBC dilutions as these are measured by different laboratory methods (photometry and laser beam dispersion, respectively). Hence;

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

( 6 )

 6 mmHg and remained elevated during the last 15 min of the infusion and onward, S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society DISCUSSION Hypoglycamia and hypovolaemia were common after the intravenous infusions of glucose in well-trained males, regardless or whether the glucose load was accompanied by exogenous insulin or a fluid volume of 1.6 litres. Blood glucose and plasma dilution regularly fell to below baseline within 30 min after the glucose infusion ended. Three hours later these alterations of body physiology were only partially restored, despite the fact that plasma insulin had returned to baseline.

  the "glucose only" experiments was to obtain a figure for volume of distribution and baseline endogenous glucose production in the present group of volunteers. Estimates of CL over time for all experiments could then be obtained by inserting those values into Eqn. 1. Such calculations indicate that the exogenous insulin doubled CL. Moreover, CL was only slightly higher during the "strong" experiments despite the marked difference in insulin dose between the two series of glucose-insulin infusions. Here, CL should be understood to express the body´s overall effectiveness to lower plasma glucose as it summarises the effects of the true CL of glucose from the extracellular fluid (ECF) space and the insulin-induced depression of the endogenous glucose production. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society virtue of osmosis. The plasma volume reduction by 5% observed after our glucose-insulin

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  4 (4.3) S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Fig. 1 The

 1 Fig.1The plasma glucose concentration (left) and plasma dilution (right) in experiments with infusion of glucose 2.5% (top), glucose 10% along with insulin (middle) and glucose 50% with insulin (bottom). Data are the mean (SD).

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Fig. 2 The

 2 Fig. 2 The change in plasma glucose from baseline versus the Hb-derived plasma dilution for 3 different periods (vertical row) of time during 3 series experiments (horizontal row) with infusion of glucose 2.5%, 10% and 50% without and with insulin. The regression line is shown whenever linear regression analys showed that the relationship is statistically significant by P<0.05. Each circle represents a single point in time during one experiment. S t a g e 2 ( a ) P O S T -P R I N T

Fig. 3 The

 3 Fig. 3 The change in plasma glucose from baseline versus the Hb-derived plasma dilution throughout the 12 experiments with the "strong" glucose-isnulin solution. Each circle represents is a single point in time during one experiment. S t a g e 2 ( a ) P O S T -P R I N T

Fig. 4 The

 4 Fig. 4 The glucose clearance (CL) over time (left ) and the linear relationship between the 10 log of the plasma insulin concentration logarithm and CL (right). Each point represents the mean of 12 experiments at a specific point in time.

Table 1 .

 1 Composition of the 3 infused solutions and excreted urine.

	Mean (SD) is given.
			"Mild" glucose-	"Strong" glucose-
		"Only" glucose	insulin infusion	insulin infusion
	Glucose infusion time (min)	60	60	120
	Volume infused (ml)*	1660 (163)	430 (42)	339 (37)
	Glucose conc. (mg/ml)	2.5	10	50
	Dose of glucose (g)	41.6 (3.6)	41.6 (3.6)	169 (18)
	Insulin infusion time (min)	0	30
	Insulin infusion rate (U/kg/hr)	0	0.1	1
	Infused insulin (U)	0	4.2 (0.4)	69.3 (5.9)
	Urine volume (ml)	1649 (529)	510 (394)	294 (189)
	Sodium infused (mmol)	115 (37)	0	0
	Sodium excretion (mmol)	85 (50)	66 (40)	34 (23)
	Potassium excretion (mmol)	23 (8)	19 (9)	9 (6)

"Flush" volumes, rescue injections of glucose and the amount of sampled blood are not included. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 May 2008 as manuscript CS20080032 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080032 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Table 2 .

 2 Pharmacokinetic parameters for glucoce when 20 ml/kg/h of glucose 2.5% was infused in 12 volunteers.

	Parameter	Mean	SD
	CL (litre/min)	0.78	0.19
	SD of estimate	0.04	0.02
	V d (litres)	7.1	3.9
	SD of estimate	0.3	0.2
	R i en (mmol/min)	3.7	0.9
	SD of estimate	0.2	0.1
	Half-life (min)	7.1	5.0

CL = clearance, V d = volume of distribution, k i en = endogenous glucose production. Half-life = 0.693 * V d / CL. _____________________________________________________________________________________

Table 3 .

 3 The change in plasma glucose from baseline and the plasma dilution for various periods of time during the glucose-insulin experiments. Data are the mean (SD).

	During	After infusion
	infusion	0-30 min	> 30 min

Acknowledgements: Nurse anaesthetists Agnetha Karlfeldt, Elinor Hahlin and Marianne

Ekström assisted during the experiments. Professors Björn Biber and Ola Winsö, Umeå University Hospital, generously helped us with facilities at ICU.

when it averaged 73 (5) mmHg (P< 0.001). The systolic pressure tended to increase at the end of the infusion but the change was not significant. Heart rate showed no trend.

The "mild" glucose-insulin infusion was followed by a 10-15% reduction of the systolic and diastolic pressures during the last 2 hours of the experiment (P< 0.006). The heart rate increased from 62 [START_REF] Sjöstrand | Intravenous hydration with a glucose 2.5% solution in type 2 diabetes[END_REF] to 67 [START_REF] Sjöstrand | Volume kinetics of 2.5% glucose solution during laparoscopic cholecystectomy[END_REF] bpm during the first hour post-infusion, after which it decreased to below baseline (to 56 [START_REF] Korvald | Myocardial substrate metabolism influences left ventricular energetics in vivo[END_REF] bpm; P< 0.0001).

During the last hour of the "strong" glucose-insulin experiments, the systolic and diastolic arterial pressures also fell by almost 10% from baseline (P< 0.05). The heart rate tended to rise post-infusion, but this change was not statistically significant.

Insulin, electrolytes and urine

The baseline serum insulin concentration was 24 ( 16) pmol/l. The maximum concentration at the end of the "mild" glucose-insulin infusion amounted to 2,325 (1,964-2,635) pmol/l (median, 25 th -75th percentiles). The baseline insulin level had been reached again at 120 min, the half-life being 27 (2) min.

In the "strong" glucose-insulin experiments, the half-life of insulin in serum was 32 (4) min and the baseline level had been reached at 210 min.

There was a statistically significant relationship between CL for glucose and log 10 of the plasma insulin concentration at the points in time when the insulin level had been assessed successfully (Fig. 4, right). Serum sodium decreased during the "only glucose" infusion (from 140.5 (2.0) to 136.6 (3.6) mmol/l; P< 0.001) but the baseline level had been restored 30 min later ended.

Serum sodium was unchanged or increased slightly during the two glucose-insulin experiments (data not shown).

Serum potassium decreased to a similar degree during the two glucose-insulin infusions, from 3.85 (0.22) to 3.55 (0.21) mmol/l (P< 0.001), after which it was slowly restored to 3.78 (0.41) mmol/l at 240 min.

The volunteers voided all or nearly all of the infused fluid during the experiments (Table 1).