L-Malate's Plasma and Excretion Profile in the Treatment of Moderate and Severe Hemorrhagic Shock in Rats

Introduction. Malate is a standard component in fluid therapy within a wide range of medical applications. To date, there are insufficient data regarding its plasma distribution, renal excretion, and metabolism after infusion. This study aimed to investigate these three aspects in a rat model of moderate and severe hemorrhagic shock (HS). Methods. Male Wistar rats were subjected to HS by dropping the mean arterial blood pressure to 25–30 mmHg (severe) and 40–45 mmHg (moderate), respectively, for 60 minutes. Subsequently, reperfusion with Ringer-saline or a malate containing crystalloid solution (7 mM, 13.6 mM, and 21 mM, resp.) was performed within 30 minutes, followed by an observation period of 150 minutes. Results. In the present experiments, malate rapidly disappeared from the blood, while only 5% of the infused malate was renally excreted. In the resuscitation interval the urinary citrate and succinate amounts significantly increased compared to control. Conclusion. Malate's half-life is between 30 and 60 minutes in both, moderate and severe HS. Thus, even under traumatic conditions malate seems to be subjected to rapid metabolism with participation of the kidneys.


Introduction
Malate, a citric acid cycle (TCA) intermediate, is a standard component in fluid therapy in a wide spectrum of medical applications. In human medicine it is used as an integrant in a concentrate to treat hypokalemia (e.g., Kalium-L-Malat 17.21%, B. Braun Melsungen), in amino acid solutions for parenteral nutrition (e.g., Aminosteril5 plus, Fresenius Kabi), in solutions, which are used to cover water, electrolyte, and energy requirements (e.g., Jonosteril5 Na100 with glucose, Fresenius Kabi), and even in pediatrics (e.g., Paediatrische Elektrolytloesung 1, Fresenius Kabi). Furthermore, malate is the central component in Jonosteril Malat (Fresenius Kabi) that is appropriate to serve as primary volume therapy in emergency medicine and fluid replacement in cases of moderate acidosis and for the maintenance of pre-, intra-, and postoperative fluid balance. In the guidelines of the German Society of Trauma, the application of crystalloid solutions like Ringer-lactate, Ringer-acetate, and Ringer-malate is recommended [1], with respect to the field of emergency medicine, to treat hemorrhage, caused by accidental trauma, and the consecutively following life-threatening consequences which represent one of the main reasons of preventable death [2][3][4]. In addition, in the guidelines of the German Society of Anaesthesiology and Intensive Care Medicine balanced solutions containing malate or acetate are recommended for the treatment of critically ill patients, whereas lactate is not [5]. Malate's positive effects in certain clinical approaches, including investigations as regards its effect on energy metabolism, cardioprotection, fibromyalgia, physical stamina, and cancer, have been shown [6][7][8][9][10][11][12][13]. Even though research regarding the treatment of hemorrhagic shock (HS) and its consequences is very scarce, the protection by administering malate is described as well [14][15][16]. In contrast, treatment of severe HS with Ringer-lactate is debatable, since earlier studies showed detrimental effects in a rat model of severe HS [17,18], whereas acetate-based balanced salt solution appeared to be predominant [19]. Certainly, acetate is reported to have vasodilatory effects resulting in cardiovascular depression in vitro as well as in vivo [20][21][22]. On the other hand, it is postulated that malate's metabolism is decelerated compared to that of acetate [23,24], but not confirmed in hemorrhagic shock.
Though the previous examinations cited above yielded encouraging results to date and malate being a standard component of intravenous fluid therapy in a wide range of medical applications, information about malate's plasma distribution and excretion profile and its metabolism after incorporation into the tissues is very scarce. Therefore, the objective of the present study was to investigate malate's plasma distribution as well as renal excretion and its metabolic fate in the experimental models of severe and moderate HS. HS was chosen because of malate's potential to protect against shockinduced damage [15], thereby becoming relevant in the preclinical and early hospital therapy of traumatic bleeding.

Animals.
Altogether, 48 male Wistar rats (420 g-500 g, age 12-14 weeks) were obtained from the central animal unit of the Essen University Hospital. Animals were kept under standardized conditions of temperature (22 ∘ C ± 1 ∘ C), humidity (55% ± 5%), and 12/12-hour light/dark cycles. They were fed ad libitum (ssniff-Spezialdiaeten, Soest, Germany) with free access to water and not fasted before the experiments.
Experiments were conducted in accordance with the standards of Annex III of the directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes [25]. The experimental protocol was reviewed and approved by the local Animal Care and Use Committee (Animal Care Center, University of Duisburg-Essen, Essen, Germany, and the district government of Duesseldorf ("North Rhine-Westphalia State Environment Agency", Recklinghausen, Germany)) with a Permit Number 84-02.04.2012.A341, G1318/12.

Anesthesia, Analgesia, and Surgical
Procedures. Anesthesia, analgesia, catheter insertion, and blood sampling were performed as described previously [19]. The rats were initially anesthetized with isoflurane (2.0% in 100% medical O 2 at 4.0 L/min) in an induction chamber. Throughout the experiment animals were kept anesthetized through a face mask (1.0-2.0% isoflurane in 100% medical O 2 at 1.0 L/min) connected to a vaporizer (Isoflurane Vet. Med. Vapor, Draeger, Luebeck, Germany) and received ketamine (50 mg/kg body weight subcutaneously) into the right chest wall for analgesia. After local lidocaine administration (5 mg/kg body weight subcutaneously), a skin-deep inguinal incision of about 2-3 cm was made, femoral vessels were freed from tissue (sparing the femoral nerve), and a Portex catheter was placed within the right femoral artery and the right femoral vein. Again after local lidocaine administration (5 mg/kg body weight subcutaneously) a median laparotomy of about 2 cm was performed to catheterize the bladder to obtain urinary samples. For the period of resuscitation, the prolonged infusion of 0.9% NaCl solution (5 mL/kg body weight × h, 37 ∘ C), to compensate intraoperative fluid depletion over surgical areas and the respiratory epithelium, was interrupted. At the end of the experiment, animals were sacrificed by resection of the heart under deep isoflurane anesthesia (4.0% isoflurane in 100% medical O 2 at 1.0 L/min).

Induction of Hemorrhagic Shock and Resuscitation
Process. The induction of hemorrhagic shock and resuscitation regimens were performed as previously described [18]. Briefly, hemorrhagic shock was induced at = 30 min by removing either 2 mL (severe shock) or 1 mL (moderate shock) blood every three minutes through the femoral artery catheter using a 2 mL syringe (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Blood from the first withdrawal was used for assessment of blood and plasma parameters via blood gas analysis. The second syringe was prefilled with either 0.2 mL (severe shock) or 0.1 mL (moderate shock) of ACD-A solution. The syringe with citrated blood was stored at 37 ∘ C and used to regulate shock severity, if needed. Blood withdrawal was continued until the mean arterial blood pressure (MAP) dropped to (1) 25-30 mmHg (severe shock) or (2) 40-45 mmHg (moderate shock); shock induction typically took about 20 min. The animals in the comparative groups all possessed a similar weight (420 to 500 g), resulting in a nearly identical volume of the shed blood. The blood volume withdrawn was about (1) 13.9 mL (severe shock) or (2) 11.5 mL (moderate shock). For the next 60 min, the MAP was retained, usually without further intervention. In some cases, small amounts (0.1-0.5 mL aliquots) of the citrated blood had to be administered, or additional small blood samples (0.5-1.0 mL aliquots) had to be withdrawn, to keep the MAP within the desired range. After the shock phase, study group-specific resuscitation fluids were infused into the femoral vein within 30 min using a syringe pump (Perfusor Secura FT, B. Braun) in a randomized and blinded manner. Experiments were continued for another 150 min or until the rat died.

Experimental
Groups. The following experimental groups were compared: (i) Severe shock/RS (shock, resuscitation with RS, six animals).
The composition of the two Ringer-based solutions with the higher and lower malate concentrations (MR7, MR21) are listed in Chemicals/Materials. The volume of the resuscitation fluids is based on the 3 : 1 rule (3 times the volume of the shed blood volume) [26].
2.6. Biomonitoring. Systolic blood pressure, diastolic blood pressure, and MAP were displayed on a monitor and documented continuously, by using the femoral artery catheter, which was connected to a pressure transducer. To keep the catheter functional RS was infused at a rate of 3 mL/h. Heart rate was determined from systolic blood pressure spikes.
Recording of MAP and heart rate was intended to monitor an adequate anesthesia and to supervise the particular blood pressure (see above). These parameters were shown elsewhere [18]. The core body temperature of all rats was monitored using a rectal sensor and was maintained around 37.3 ∘ C to 37.6 ∘ C during the whole experiment via an underlying thermostat-controlled operating table and by covering the animal with aluminum foil. Peripheral oxygen saturation was recorded continuously using a pulse oximeter (OxiCliq A, Nellcor, Boulder, CO, USA) placed at the left hind limb. All parameters were recorded periodically every 10 min (exception at = 125, 5 minutes in resuscitation).

Assessment of Blood and Plasma Parameters.
Using a 2 mL syringe containing 80 IU electrolyte-balanced heparin, blood samples (0.5 mL, except of shock induction: 2 mL) were taken from the femoral artery at For each blood sampling the animals were substituted with a 0.5 mL bolus of 0.9% NaCl solution via the femoral artery compensating the lost volume and keeping the catheter functional. In order to obtain plasma for the assessment of marker enzyme activities, blood was centrifuged at 4000 ×g for 15 min at room temperature. The gained plasma was aliquoted (50 L) and stored at −80 ∘ C until further examination. The plasma activity of lactate dehydrogenase (LDH) as a general marker for cell injury, creatine kinase (CK) activity as a marker for muscle cell injury, aspartate transaminase (ASAT) and alanine transaminase (ALAT) activities as marker for liver injury, and creatinine and urea as markers for renal injury were determined with a fully automated clinical chemistry analyzer (respons920, DiaSysDiagnostic Systems GmbH, Holzheim, Germany).  Regarding parameters of organ injury, severe and moderate hemorrhagic shock partly induced distinct increases in enzyme activities (LDH, CK, ASAT, and ALAT, not shown). In some part, animals resuscitated with MR7, MR13.6, or MR21 showed significantly reduced enzyme activities during the observation period compared to the RS-control group, independent of the administered malate concentration.  (Figures 1(a) and 1(b)). In the moderate hemorrhagic shock group, during the shock induction and shock phase, the plasma malate concentrations only slightly increased but did not exceed initial values (Figure 1(a)). The plasma malate concentrations rose noticeably to 0.19 mM (MR7), 0.57 mM (MR13.6), and 0.98 mM (MR21), respectively, during the resuscitation period depending on the administered malate amount. At the end of the experiment ( = 300 min) plasma malate concentrations were returned to initial values of <0.15 mM. In the severe shock group, values rose heterogeneously during the shock induction and shock phase (Figure 1(b)). At the end of the resuscitation phase, malate concentrations increased, depending on the infused malate amount, to 0.75 mM (MR7), 1.57 mM (MR13.6), and 2.30 mM (MR21), respectively. At the end of the experiment ( = 180 min, determined by the first animal's death in the RS-control group) malate levels decreased to 0.43 mM (MR7), 0.73 mM (MR13.6), and 1.78 mM (MR21). Values in the RS-control group stayed nearly constant over the whole experimental time.

Effects of RS
In the moderate hemorrhagic shock groups basal plasma citrate concentrations were below 0.25 mM (Figure 2 3(a) and 3(b)). During the shock induction phase it clearly decreased, independent of the particular shock depth, to about 110 L. In the moderate hemorrhagic shock groups, urine excretion clearly increased to 621 L (MR7), 710 L (MR13.6), 633 L (MR21), and 689 L (RS) during the resuscitation interval ( = 120-150 min; Figure 3(a)). In the two subsequent observation intervals ( = 180-240 min and = 240-300 min) uroflow slightly decreased, reaching almost initial levels. In contrast, in the severe hemorrhagic shock groups urine excretion stayed nearly constant at shock induction levels during the remaining period of the experimental time (Figure 3(b)).
Renal excretion of malate, citrate, and succinate was only measured under conditions of moderate hemorrhagic shock, due to the small urinary sample volumes in the severe shock groups.
Urinary succinate concentration rose significantly in the experimental groups receiving MR21 during the resuscitation interval (Figure 6(a)). Only in the resuscitation time interval ( = 120-150 min) did the renal succinate excretion increase slightly to 1.4 mol (MR7) and 3 mol (MR13.6) and increase significantly to 6 mol (MR21), while in the last interval ( = 240-300 min) again initial excretion levels of <1 mol were reached (Figure 6(b)).

Discussion
Although malate is a standard component in fluid therapy, information is lacking about its plasma distribution, renal excretion, and metabolism after administration. In the present experiments, malate rapidly disappeared from the blood. Since only a minor amount of the infused malate was eliminated via the kidneys, the vast majority was obviously metabolized.
In moderate HS, plasma malate concentrations sharply increased in a dose-dependent manner at the end of the resuscitation interval but decreased to baseline values within 2.5 hours (Figure 1(a)). Assuming that malate's plasma clearance is nearly linear, a half-life of about 1 hour can be derived. On the other hand, most drugs that are administered intravenously are proportionally eliminated to their plasma concentration (first-order kinetics) [27,28]. In turn, that means a half-life of 1 hour is overestimated. This is also supported by the results of malate's plasma clearance in severe HS. Even under these conditions (expecting a more decelerated metabolism compared to moderate HS), plasma malate concentrations showed a steep decline, partly up to 54%, already 30 min after resuscitation had ended (Figure 1(b)). These findings suggest that the plasma clearance of malate should be less than 1 hour. Moreover, in moderate HS only 5% of the infused malate was renally excreted, whereupon the vast majority had already been excreted within the reperfusion interval ( Figure 4). In severe HS, an increased renal excretion is virtually excluded, since uroflow is excessively impaired (Figure 3(b)). Therefore, based on the present results, it can be concluded that malate's half-life is between 30 min and 60 min under hemorrhagic conditions.  [29]. Furthermore, malate's rapid removal from the blood as well as its excretion profile indicates uptake into the cells with subsequent metabolism (see below). Apart from the liver [30], skeletal muscle seems to be an important malate consumer, since studies showed malate's positive impact in treating fibromyalgia [31] and its influence on the exercising muscle [6], both probably related to an improved energy metabolism. An ubiquitous metabolism is further supported by the present results. Malate rapidly disappeared from the blood (see above) and the increases in the absolute urinary amounts of the two TCA intermediates citrate and succinate suggest an additional participation of the kidneys in malate's metabolism after infusion. In line, increased renal excretion of citrate following malate has also been reported in healthy test subjects [32][33][34].
Of the metabolizable anions usually used in fluid therapy, acetate's plasma clearance after infusion follows first-order kinetics, being absorbed within minutes [29,35]. Acetate has been reported to be renally excreted from less than 10% (in healthy volunteers) [36] up to 40% [37] under the special condition of acetate's application right into the left renal artery in an experimental dog model. Acetate can be metabolized along with the liver by the heart, adipose tissue, kidneys, and muscle [38]. Along with malate and acetate, lactate is a common component in infusion solutions but is metabolized mainly in the liver [39]. This fact presupposes a sufficient liver function, which is typically impaired in terms of hemorrhage and hypovolemic conditions, respectively, leading to adverse effects of the accumulated endogenous lactate and the given lactate during resuscitation (e.g., exacerbated lactic acidosis, inhibition of glycolysis) [17,18,[40][41][42]. The adverse effects are additionally fostered by nonexistent renal elimination of lactate [43].
Previous experiments resulted in malate's predominance compared to Ringer's lactate and Ringer's acetate in resuscitation of hemorrhagic shock independent of the shock depth, regarding, for example, survival [15]. In line with these results, regarding malate's protective properties, we could show that MR increased the median survival time after severe hemorrhagic shock, compared to pure RS, independently of the administered malate concentration. The prolonged survival of shocked animals that were resuscitated with MR may be due to a less severe organ injury, which is indicated by the reduced release of intracellular enzymes. In turn, the lower tissue damage could be closely associated with the nearly ubiquitous metabolism of malate, thereby being rapidly available, for example, as precursor for adenosine triphosphate production, for various organs.

Conclusion
In conclusion, in the present experiments malate showed delayed plasma half-life compared to acetate's literature references. Nevertheless, the still rapid disappearance from the circulation and the marginal renal excretion during the experimental time is of high interest, because the administered malate dosage is virtually completely available to exert its therapeutic actions. Malate's protective effect in the treatment of HS is probably related to its rapid incorporation into the tissues and thereby, for example, being rapidly available as a precursor for adenosine triphosphate production. The nearly ubiquitous metabolism of malate may prevent a potential overload of specific tissues thus giving it a clear advantage over lactate-containing infusion solutions.