Clinical research
Advantage of adjunct metformin and insulin therapy in the management of glycemia in critically ill patients. Evidence for nonoccurrence of lactic acidosis and needing to parenteral metformin

Introduction
Critical illness (CI) is associated with alterations in neuroendocrine and immune systems [1, 2]. Disturbances in immunoneuroendocrine axis lead to subsequent alterations in carbohydrate and lipid metabolisms provoking systemic inflammatory responses [2, 3]. Stress-induced hyperglycemia occurs due to multiple pathogenic mechanisms and associate with development of adverse events and poor prognostic outcomes in severe illness [4]. Today it is well known that glycemic control and intensive insulin therapy decrease morbidity and mortality rates and improve survival of critically ill patients (CIPs) [5-7]. Although several insulin protocols have been designed and applied in different setting of CIPs [5, 6, 8], they have carried the highest risk of hypoglycemia [9]. Intensive insulin therapy promotes entry of potassium and magnesium from the extra-cellular to the intracellular compartment and possibly leading to hypokalemia and hypomagnesaemia [10, 11]. Reductions in potassium and magnesium levels promote insulin resistance [12-14] and higher blood glucose level (BGL). Thus, administration of more insulin is inevitable that initiates the vicious cycle with adverse outcomes. In this regard, blood levels of potassium and magnesium as well as BGL must be closely monitored during insulin therapy [15]. Insulin sensitizing agents are prescribed widely in outpatients for those individual with insulin resistance such as many of the type 2 diabetics [16]. Metformin as an insulin-sensitizing agent may overcome insulin resistance in CI but it has been suspected for lactic acidosis (LA) [17]. Several studies have demonstrated that metformin, per se, does not promote LA and this phenomenon is coincidental with other underlying CIs [18, 19]. Except two trials in small population of the burned patients [20, 21], the efficacy of metformin in glycemic control in CIPs has not been studied. In the present study, we investigated the effectiveness and safety of metformin in the glycemic control of CI traumatized patients by measuring the blood glucose, lactate and pH values of the patients, in addition to their insulin requirements when insulin coadministered with metformin.
Material and methods

Study population
The study has been reviewed and approved by the Institutional Review Board (IRB). A written informed consent was obtained from the closest patient’s relative in the hospital. All adult multiple traumatized patients who were admitted to the intensive care unit (ICU) and diagnosed with both systemic inflammatory response syndrome (SIRS) and hyperglycemia (Table I) were eligible to be enrolled into study. Eleven patients were included in each therapeutic arm on the basis of a pilot study. Of 33 patients who were enrolled in the study, 4 were excluded due to severe hemodynamic instabilities or developing the excluding criteria (Table I) during the first 12 hours. Baseline characteristics of the patients are demonstrated in Table II.
Study design
Study was carried out between July 2005 and September 2006 in a randomized open label clinical trial fashion, in the medical-surgical ICU of a University hospital. On admission to ICU, those patients who met the inclusion criteria were randomized to receive one of the following protocols for the period of one week. Protocols included intensive insulin therapy (A), metformin monotherapy (B), and intensive insulin therapy in combination with metformin (C). Protocols were instituted to maintain BGL between 80-120 mg/dl as target level. In protocol A, insulin was administered through continuous intravenous infusion (50 IU insulin regular in 50 ml of 0.9% sodium chloride). The infusion rate was started according to admission BGL and adjusted to a rational algorithm (Table III). BGL was measured at bed every 2 hours. During infusion of insulin with rate of higher than 5 IU/hour, BGL was measured every hour. Whenever BGL reached below 120 mg/dl, the infusion of insulin was stopped. In protocol B, patients received 1000 mg of metformin every 12 hours. BGL and arterial blood lactate was measured every 6 hours at bed. In both protocols B and C, metformin was discontinued if lactate level was more than 4.5 mmol/l, or if increased more than 2 mmol/l form the previous lactate level, or if bicarbonate reached lesser than 13 mEq/l, or if pH reached lesser than 7.3, or if two consecutive serum creatinine levels were higher than 1.2 mg/dl, and if maximum arterial pressure reached lesser than 70 mm Hg during the intervention. In protocol C, similar to protocol A, infusion of insulin started according to admission BGL. Adjustment of insulin dose was done on BGL fluctuations. In addition, patients received 1000 mg metformin orally every 12 hours. Blood glucose and lactate levels were measured every 2 and 6 hours respectively.
Pharmacokinetic study of metformin
A rapid and specific ion pair HPLC method was designed for determination of plasma metformin concentration. A Knauer HPLC system (Germany) was used consisting a pump (K-1001) and a UV detector (K 2600). The analytical column was Teknokroma Lichrospher 100 CN (5 µm 4.6 × 250 mm). The mobile phase consisted of 30% acetonitrile and 70% sodium lauryl sulfate (SLS) 0.1% aqueous solution. The method was validated for selectivity, accuracy, precision, and calibration curve on the basis of FDA guideline for validation of bioanalytical method [22]. Blood samples were obtained at 7 time points: before administration of the drug, and at 24, 26, 28, 30, 33, 36 hours after the first dose. Patients’ blood samples were collected in heparinized glass tubes and centrifuged. Subsequently, separated plasmas were frozen at –20°C until analysis [23, 24].
Calculation of pharmacokinetic parameters of metformin
The pharmacokinetics of metformin was determined by noncompartmental analysis. The maximum plasma concentrations (Cmax) was recorded as observed. The elimination rate constant (Ke) was estimated as the absolute value of the slope of a least-square linear regression of the terminal phase of the logarithmic plasma concentration-time curve. The plasma elimination half-life (t1/2e) was calculated as 0.693/Ke. The area under the plasma concentration-time curves (AUC) from time zero to the infinite time AUC(0-12) was calculated by trapezoidal rule during the dosing interval. Apparent volume of distribution and total clearance were calculated as D° D° Vd/F = –––––––– , Cl/F = ––––– , respectively. Ke.[AUC] [AUC]
Data collection
For calculating acute physiology and chronic health evaluation II (APACHE II) score [25], arterial blood gases, electrolytes, and complete blood counts were determined daily, and blood pressure, heart rate, respiratory rate, body temperature, pulmonary indices, and hemodynamic profiles were checked and recorded every 2 hours and mean daily values were calculated.
Statistical analysis
Daily outcome variables were compared between each pair of groups. Each group was compared with two others by use of independent samples t-test. The normality of distribution of variables was examined with Kolmogrov-Smirnov test. Comparisons between admission values of BGL, blood lactate level and first day APACHE II score and mean values in the course of the study in each group were performed by paired sample t-test. Chi-square test was performed for analysis of mortality. Data are mean ± SD unless otherwise stated. Significant level was defined as P<0.05.
Results

Mortality
Four patients in group A, 2 in group B, and 3 in group C died during the intensive care. Difference in the mortality rate of these groups was not statistically significant (P>0.05).
Glycemic control by protocols A, B, and C
By applying of all protocols, the initial BGL decreased. Admission time BGL in protocol A, B, and C were 191±28, 189±35, and 192±28 mg/dl respectively. Following institution of protocols (A, B, and C), these values reduced and mean weekly BGL reached 122±9, 131±17, and 121±7 mg/dl, respectively (Figure 1). No significant difference in the control of glycemia was observed between three protocols. Comparison of initial BGL and mean weekly values showed a significant reduction in group A (P=0.02) and B (P=0.003) but the 36% reduction in admission BGL of group C was not statistically significant (P=0.17).
Insulin requirement
During the course of the study, mean daily insulin requirements of patients in the protocols A and C were 19.5 and 12.7 IU/day. A 34% reduction in mean daily insulin infusion dose was observed in protocol C as compared to protocol A but it was not statistically significant (Figure 2).
Lactate monitoring
Although patients had some fluctuations in their blood lactate levels, there was no severe hyperlactatemia (blood lactate level >5 mmol/l). The admission lactate values were 2.7±0.4 and 2.6±1.2 mmol/l in groups B and C that reached to 2.4±0.5 and 2.3±0.15 mmol/l, respectively. These fluctuations in blood lactate levels were not significant within each groups [P(B)=0.45 and P(C)=0.79] and there was no significant difference between two groups (P=0.66) (Figure 3).
Acid base balance in protocols A, B, and C
No significant difference was observed between mean daily pH values of the patients in protocols A, B, and C. All the calculated comparison P values were >0.9. There was no pH <7.3. pH values had tiny fluctuations in normal range.
Effects of protocols on APACHE II scoring
No significant difference was observed in weekly APACHE II score between the protocols A, B, and C and within the patients (Table IV) but the reduction in basal APACHE II score in group B was significant (P=0.005).
Pharmacokinetic profile
Pharmacokinetic parameters of metformin including Cmax, Ke, T1/2e, AUC(0-12), Vd/F, and Cl/F are shown in Table V. The Ke and T1/2e values were in the range of normal values of healthy or outpatient diabetic subjects. Cmax and AUC(0-12) levels were lower than normal values. The Vd/F and Cl/F values were higher than normal ranges.
Correlation between pharmacokinetic and clinical parameters
No significant correlation was observed between plasma drug concentration and reduction of blood glucose or blood lactate levels, or blood pH values (Table VI).
Discussion
Major clinical outcomes of the present study Results of the present study indicate that three protocols successfully reduce initial BGL. Although the present mean plasma metformin level was lesser than its reported therapeutic levels in outpatients (0.5-1 mg/l in the fasting state and 1-2 mg/l after meals) [26], the patients’ BGL reduced and their insulin requirements decreased. Despite a few mild increase in BGL of some patients that might be related to medical interventions or instability of patients in acute and catabolic phase of critical illness, both protocols of A and C reduced BGL to the target points (80-120 mg/dl) during the first 48 hours. Nevertheless, temporal reduction of BGL in group C was not statistically significant possibly due to scarce number of the patients. Comparison of insulin requirements between protocols A and C show a 34% reduction in average daily insulin dose in protocol C; although this reduction was not statistically significant (P=0.21), the insulin requirement reduced in combination with metformin indicating potential of metformin to overcome insulin resistance. Metformin monotherapy controlled hyperglycemia and improved APACHE II that adequately confirm its benefit in CIPs. Nevertheless, it is suggested to conduct such investigations in larger population to reach more extendable and reliable results.
Current insights in glycemic control of CIPs
It has been demonstrated that intensive insulin therapy in the absence of euglycemia has no significant effect on improvement of the patients’ outcome [27, 28]. Likewise, it has been strongly suggested that metabolic control as a result of normoglycemia rather than insulin infused dose is related to beneficial effects of intensive insulin therapy. Thus, although insulin itself has plenty of beneficial metabolic and non-metabolic effects in CIPs [4, 7, 29, 30], it seems intensive glycemic control has a pivotal role in improvement of outcomes rather than intensive insulin therapy alone [31]. Insulin resistance and inflammatory processes that are characterized by increased proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) [32, 33], have crucial roles in the pathogenesis of stress-induced hyperglycemia [4, 34, 35]. Current approach to control this metabolic disorder is intensive insulin therapy that associates with some complications. Insulin is one of the five highest risk medications in the inpatient setting. To avoid insulin adverse effects, tight glycemic control, and monitoring of potassium and magnesium levels is inevitable that accompany with higher nursing workload [15]. The current approach to manage metabolic syndrome and type 2 diabetes mellitus in outpatients with insulin resistance is administration of insulin sensitizing agents like metformin [16]. This medication enhances insulin’s actions [36] and has anti inflammatory properties which prevent initiation and progression of inflammatory processes [4, 26, 37, 38]. Thus, regarding the significant role of proinflammatory cytokines and oxidative stress mediators in pathogenesis and prognosis of many chronic and acute diseases [39-43], administration of such medications could be a novel therapeutic approach to prevent and control hyperglycemia in CIPs. However, safety, efficacy, and pharmacokinetic of these medication are the matter of concerns in ICU patients.
Evaluation of key pharmacokinetic indices of metformin in CIPs
Previous studies in healthy volunteers and diabetic outpatients demonstrated that there is a proportional relationship between plasma maximum metformin concentration and administered dose [44]. Our study showed that despite the higher administered dose (1000 mg BD), Cmax levels were lower than Cmax levels in other single oral dose studies with lesser dosage size [44], and the AUC is small indicating a defect in the absorption of drug. This defect might back to existence of gastrointestinal (GI) abnormalities such as organ hypoperfusion and hypomotility during CIs [45, 46]. The apparent volume of distribution and total clearance of metformin in the present study were higher than reported values for healthy subjects or diabetic patients [44]. As a matter of fact, fluids shift to interstitium and thus administration of crystalloids or colloids fluids and alteration in plasma proteins could not be suspected. In addition, metformin per se has a large volume of distribution and its protein binding is negligible [44, 47]. Therefore, the most probable reason for these high volumes of distribution and total clearance values is low F value (F is the fraction of drug absorbed) which is mainly related to the considerable GI abnormalities. Potential alterations in oral bioavailability makes the intravenous rout of administration as a generally preference in CIPs [38]. With due attention to low bioavailability of metformin in healthy subjects or outpatients (40 to 60%) [44, 47] and lower bioavailability in CIPs, the intravenous administration should be tried to evaluate exact effects of drug in higher plasma concentration. Some investigations concluded that circulating metformin concentration correlates with plasma glucose levels in outpatients [44] that is not supported by the present findings since no significant association between AUC and reduction of BGL was observed. The explanation is that pharmacodynamic profile of the drug may differ in CI and some unproven subcellular mechanisms may orchestrate its pharmacologic effects in stressful situations.
Safety of metformin in CIPs
Except nausea and vomiting, other adverse effects of metformin such as vitamin B12 deficiency are not matter of concern in CIPs because they do not occur in a short period of time [48]. The present data did not show even GI nausea or vomiting. Despite the probable differences between the rate and extent of absorption of the drug in different days, no correlation was observed among AUC and intraday degree of difference values of lactate. The present data indicated no correlation between plasma metformin concentration and blood lactate or pH levels. Although hyperlactatemia (>2 mmol/l) was frequently seen in the study patients [median (interquartile range) was 2.4 (2.1-2.7)], metformin did not induce lactic acidosis (>5 mmol/l and pH <7.3). Lactic acidosis is claimed as a limitation in the use of metformin in CIPs [49, 50] but it is too difficult to differentiate hyperlactatemia as a common sequel of metabolic alterations in severe illness [51] with that of metformin. Some investigations confirmed coincidental rather than causal association between metformin and lactic acidosis in outpatients [19, 52]. On the other hand, other investigations found no obvious relationship between metformin accumulation and rate of lactic acidosis or the associated mortality in patients with precipitating conditions who received therapeutic doses of the drug [53]. Likewise, some studies have shown that underlying hemodynamic conditions are the main determinant of hyperlactatemia and there is no association between metformin accumulation and blood lactate levels [18, 54]. Since the incidence of lactic acidosis is rare in metformin therapy [17-19], to attain more convincing results, more studies should be conducted in larger number of patients.
Efficacy of metformin in acute care medicine
Some investigators have reported no acute effect of metformin on production of hepatic glucose or peripheral glucose disposal [55]. In contrast, there are reports indicating benefit of acute metformin in insulin resistant patients [56-59] that is supported by the present study. Fortunately, no complications such as hypoglycemia, hypokalemia, and hypomagnesaemia that commonly happen in insulin therapy [36], were observed in the present study. Taking collectively, this preliminary study suggests the benefit of combination therapy of hyperglycemic CIPs with metformin and insulin but remains to be confirmed by more experimental and clinical investigations with larger sample number in different types of CIPs. Of course, it should not be forgotten that CIPs are under influence of various stresses that can disturb both kinetic and dynamic of drugs used in ICU [60, 61] seeming the source of controversies among different reports.
Acknowledgments
This study was supported by a grant from Research Council of TUMS. Authors wish to thank helps of A. Meysamie, M.R. Khajavi, F. Mohammadzadeh, and H. Moalemi.
References
1. Mizock BA. Alterations in fuel metabolism in critical illness: hyperglycaemia. Best Pract Res Clin Endocrinol Metab 2001; 15: 533-51. 2. Beishuizen A, Thijs LG. The immunoneuroendocrine axis in critical illness: beneficial adaptation or neuroendocrine exhaustion? Curr Opin Crit Care 2004; 10: 461-7. 3. Robertson CM, Coopersmith CM. The systemic inflammatory response syndrome. Microbes Infect 2006; 8: 1382-9. 4. Kajbaf F, Mojtahedzadeh M, Abdollahi M. Mechanisms underlying stress-induced hyperglycemia in critically ill patients. Therapy 2007; 4: 97-106. 5. Van den Berghe G, Wouters P, Weekers F, et al. Intensive insulin therapy in the critically ill patients. N Engl J Med 2001; 345: 1359-67. 6. Krinsley JS. Effect of an intensive glucose management protocol on the mortality of critically ill adult patients. Mayo Clin Proc 2004; 79: 992-1000. 7. Van den Berghe G. How does blood glucose control with insulin save lives in intensive care? J Clin Invest 2004; 114: 1187-95. 8. Brown G, Dodek P. Intravenous insulin nomogram improves blood glucose control in the critically ill. Crit Care Med 2001; 29: 1714-9. 9. Van den Berghe G, Wilmer A, Milants I, et al. Intensive insulin therapy in mixed medical/surgical intensive care units: benefit versus harm. Diabetes 2006; 55: 3151-9. 10. Gennari FJ. Hypokalemia. N Engl J Med 1998; 339: 451-8. 11. Delva P, Degan M, Trettene M, Lechi A. Insulin and glucose mediate opposite intracellular ionized magnesium variations in human lymphocytes. J Endocrinol 2006; 190: 711-8. 12. Nadler JL, Buchanan T, Natarajan R, Antonipillai I, Bergman R, Rude R. Magnesium deficiency produces insulin resistance and increased thromboxane synthesis. Hypertension 1993; 21: 1024-9. 13. Huerta MG, Roemmich JN, Kington ML, et al. Magnesium deficiency is associated with insulin resistance in obese children. Diabetes Care 2005; 28: 1175-81. 14. Choi CS, Thompson CB, Leong PK, McDonough AA, Youn JH. Short-term K(+) deprivation provokes insulin resistance of cellular K(+) uptake revealed with the K(+) clamp. Am J Physiol Renal Physiol 2001; 280: F95-F102. 15. ACE/ADA Task Force on Inpatient Diabetes. American College of Endocrinology and American Diabetes Association Consensus statement on inpatient diabetes and glycemic control: a call to action. Diabetes Care 2006; 29: 1955-62. 16. Krentz AJ, Bailey CJ. Oral antidiabetic agents: current role in type 2 diabetes mellitus. Drugs 2005; 65: 385-411. 17. Wiholm BE, Myrhed M. Metformin-associated lactic acidosis 1977-1991. Eur J Clin Pharmacol 1993; 44: 589-91. 18. Lalau JD, Race JM. Lactic acidosis in metformin therapy. Drugs 1999; 58 (Suppl. 1): 55-60. 19. Stades AM, Heikens JT, Erkelens DW, Holleman F, Hoekstra JB. Metformin and lactic acidosis: cause or coincidence? A review of case reports. J Intern Med 2004; 255: 179-87. 20. Gore DC, Wolf SE, Herndon DN, Wolfe RR. Metformin blunts stress-induced hyperglycemia after thermal injury. J Trauma 2003; 54: 555-61. 21. Gore DC, Herndon DN, Wolfe RR. Comparison of peripheral metabolic effects of insulin and metformin following severe burn injury. J Trauma 2005; 59: 316-22. 22. Guidance for Industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), May 2001. http: //www.fda.gov/cder/guidance. 23. Amini H, Ahmadiani A, Gazerani P. Determination of metformin in human plasma by high-performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 2005; 824: 319-22. 24. Zarghi A, Foroutan SM, Shafaati A, Khoddam A. Rapid determination of metformin in human plasma using ion-pair HPLC. J Pharm Biomed Anal 2003; 31: 197-200. 25. Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: a severity of disease classification system. Crit Care Med 1985; 13: 818-29. 26. Caballero AE, Delgado A, Aguilar-Salinas CA, et al. The differential effects of metformin on markers of endothelial activation and inflammation in subjects with impaired glucose tolerance: a placebo-controlled, randomized clinical trial. J Clin Endocrinol Metab 2004; 89: 3943-8. 27. Malmberg K, Rydén L, Wedel H, et al.; DIGAMI 2 Investigators. Intense metabolic control by means of insulin in patients with diabetes mellitus and acute myocardial infarction (DIGAMI 2): effects on mortality and morbidity. Eur Heart J 2005; 26: 650-61. 28. Mehta SR, Yusuf S, Díaz R, et al.; CREATE-ECLA Trial Group Investigators. Effect of glucose-insulin-potassium infusion on mortality in patients with acute ST-segment elevation myocardial infarction: the CREATE-ECLA randomized controlled trial. JAMA 2005; 293: 437-46. 29. Vanhorebeek I, Langouche L, Van den Berghe G. Glycemic and nonglycemic effects of insulin: how do they contribute to a better outcome of critical illness? Curr Opin Crit Care 2005; 11: 304-11. 30. Andreelli F, Jacquier D, Troy S. Molecular aspects of insulin therapy in critically ill patients. Curr Opin Clin Nutr Metab Care 2006; 9: 124-30. 31. Van den Berghe G, Wouters PJ, Bouillon R, et al. Outcome benefit of intensive insulin therapy in the critically ill: Insulin dose versus glycemic control. Crit Care Med 2003; 31: 359-66. 32. Hadidi E, Mojtahedzadeh M, Paknejad MH, et al. Alterations of blood IL-8, TGF-beta1 and nitric oxide levels in relation to blood cells in patients with acute brain injury. Therapy 2006; 3: 413-9. 33. Salari P, Mojtahedzadeh M, Najafi A, et al. Comparison of the effect of aminophylline and low PEEP vs. high PEEP on EGF concentration in critically ill patients with ALI/ARDS. J Clin Pharm Ther 2005; 30: 139-44. 34. Dandona P, Mohanty P, Chaudhuri A, Garg R, Aljada A. Insulin infusion in acute illness. J Clin Invest 2005; 115: 2069-72. 35. Robinson LE, van Soeren MH. Insulin resistance and hyperglycemia in critical illness: role of insulin in glycemic control. AACN Clin Issues 2004; 5: 45-62. 36. Kirpichnikov D, McFarlane SI, Sowers JR. Metformin: an update. Ann Intern Med 2002; 137: 25-33. 37. Hattori Y, Suzuki K, Hattori S, Kasai K. Metformin inhibits cytokine-induced nuclear factor kappaB activation via AMP-activated protein kinase activation in vascular endothelial cells. Hypertension 2006; 47: 1183-8. 38. Rahimi R, Nikfar S, Larijani B, Abdollahi M. A review on the role of antioxidants in the management of diabetes and its complications. Biomed Pharmacother 2005; 59: 365-73. 39. Soltan-Sharifi MS, Mojtahedzadeh M, Najafi A, et al. Improvement by N-acetylcysteine of acute respiratory distress syndrome through increasing intracellular glutathione, and extracellular thiol molecules and anti-oxidant power: evidence for underlying toxicological mechanisms. Hum Exp Toxicol 2007; 26: 697-703. 40. Larijani B, Afshari M, Astanehi-Asghari F, et al. Effect of short-term carvedilol therapy on salivary and plasma oxidative stress parameters and plasma glucose level in type II diabetes. Therapy 2006; 3: 119-23. 41. Vazin A, Mojtahedzadeh M, Najafi A, Khalilzadeh A, Abdollahi M. Relationship between duration, fatality rate and severity of disease and serum epidermal growth factor in human acute lung injury. Therapy 2005; 2: 255-9. 42. Salari P, Mojtahedzadeh M, Abdollahi M. Influence of serum epidermal growth factor on mechanical ventilation and survival in patients with acute respiratory distress syndrome. Therapy 2005; 2: 393-8. 43. Radfar M, Larijani B, Hadjibabaie M, Rajabipour B, Mojtahedi A, Abdollahi M. Effects of pentoxifylline on oxidative stress and levels of EGF and NO in blood of diabetic type-2 patients; a randomized, double-blind placebo-controlled clinical trial. Biomed Pharmacother 2005; 59: 302-6. 44. Scheen AJ. Clinical pharmacokinetics of metformin. Clin Pharmacokinet 1996; 30: 359-71. 45. Power BM, Forbes AM, van Heerden PV, Ilett KF. Pharmacokinetics of drugs used in critically ill adults. Clin Pharmacokinet 1998; 34: 25-56. 46. Boucher BA, Wood GC, Swanson JM. Pharmacokinetic changes in critical illness. Crit Care Clin 2006; 22: 255-71. 47. Marchetti P, Giannarelli R, di Carlo A, Navalesi R. Pharmacokinetic optimisation of oral hypoglycaemic therapy. Clin Pharmacokinet 1991; 21: 308-17. 48. Ting RZ, Szeto CC, Chan MH, Ma KK, Chow KM. Risk factors of vitamin B(12) deficiency in patients receiving metformin. Arch Intern Med 2006; 166: 1975-9. 49. Clement S, Braithwaite SS, Magee MF, et al; American Diabetes Association Diabetes in Hospitals Writing Committee. Management of diabetes and hyperglycemia in hospitals. Diabetes Care 2004; 27: 553-91. 50. Owen MR, Doran E, Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 2000; 348: 607-14. 51. Valenza F, Aletti G, Fossali T, et al. Lactate as a marker of energy failure in critically ill patients: hypothesis. Crit Care 2005; 9: 588-93. 52. Salpeter SR, Greyber E, Pasternak GA, Salpeter EE. Risk of fatal and nonfatal lactic acidosis with metformin use in type 2 diabetes mellitus: systematic review and meta-analysis. Arch Intern Med 2003; 163: 2594-602. 53. Lalau JD, Race JM. Lactic acidosis in metformin therapy: searching for a link with metformin in reports of ‘metformin-associated lactic acidosis’. Diabetes Obes Metab 2001; 3: 195-201. 54. Lalau JD, Mourlhon C, Bergeret A, Lacroix C. Consequences of metformin intoxication. Diabetes Care 1998; 21: 2036-7. 55. Sum CF, Webster JM, Johnson AB, Catalano C, Cooper BG, Taylor R. The effect of intravenous metformin on glucose metabolism during hyperglycemia in type 2 diabetes. Diabet Med 1992; 9: 61-5. 56. Iannello S, Camuto M, Cavaleri A, et al. Effects of short-term metformin treatment on insulin sensitivity of blood glucose and free fatty acids. Diabetes Obes Metab 2004; 6: 8-15. 57. Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001; 108: 1167-74. 58. Bayrak A, Terbell H, Urwitz-Lane R, Mor E, Stanczyk FZ, Paulson RJ. Acute effects of metformin therapy include improvement of insulin resistance and ovarian morphology. Fertil Steril 2007; 87: 870-5. 59. Perriello G, Misericordia P, Volpi E, et al. Acute antihyperglycemic mechanisms of metformin in NIDDM. Evidence for suppression of lipid oxidation and hepatic glucose production. Diabetes 1994; 43: 920-8. 60. Mojtahedzadeh M, Vazin A, Najafi A, Khalilzadeh A, Abdollahi M. The effect of furosemide infusion on serum epidermal growth factor concentration after acute lung injury. J Infus Nurs 2005; 28: 188-93. 61. Hadidi E, Mojtahedzadeh M, Rouini MR, et al. The evaluation of the possible effect of positive end expiratory pressure (PEEP) on pharmacokinetics of phenytoin in patients with acute brain injury under mechanical ventilation. DARU 2005; 13: 74-81.