Biguanides and lactic acidosis:unraveling the complex relationship

Biguanides,such as metformin,have long been established as frontline medications for the management of type 2 diabetes due to their glucose-lowering effects and favorable safety profiles.However,concerns regarding the risk of lactic acidosis associated with biguanide use have sparked considerable debate and scrutiny.This research article aims to provide a comprehensive analysis of the relationship between biguanides,particularly metformin,and lactic acidosis.We delve into the underlying mechanisms,epidemiological evidence,risk factors,clinical manifestations,diagnostic considerations,and management strategies related to biguanide-induced lactic acidosis.Additionally,we explore recent research developments,controversies,and future directions in this critical area of pharmacovigilance and clinical practice.

Susceptibility of dairy cows to subacute ruminal acidosis is reflected in both prepartum and postpartum bacteria as well as odd-and branched-chain fatty acids in feces

Background:The transition period is a challenging period for high-producing dairy cattle.Cows in early lactation are considered as a group at risk of subacute ruminal acidosis(SARA).Variability in SARA susceptibility in early lactation is hypothesized to be reflected in fecal characteristics such as fecal pH,dry matter content,volatile and odd-and branched-chain fatty acids(VFA and OBCFA,respectively),as well as fecal microbiota.This was investigated with 38 periparturient dairy cows,which were classified into four groups differing in median and mean time of reticular pH below 6 as well as area under the curve of pH below 6.Furthermore,we investigated whether fecal differences were already obvious during a period prior to the SARA risk(prepartum).Results:Variation in reticular pH during a 3-week postpartum period was not associated with differences in fecal pH and VFA concentration.In the postpartum period,the copy number of fecal bacteria and methanogens of unsusceptible(UN)cows was higher than moderately susceptible(MS)or susceptible(SU)cows,while the genera Ruminococcus and Prevotellacea_UCG-001 were proportionally less abundant in UN compared with SU cows.Nevertheless,only a minor reduction was observed in iso-BCFA proportions in fecal fatty acids of SU cows,particularly iso-C15:0and iso-C16:0,compared with UN cows.Consistent with the bacterial changes postpartum,the lower abundance of Ruminococcus was already observed in the prepartum fecal bacterial communities of UN cows,whereas Lachnospiraceae_UCG-001 was increased.Nevertheless,no differences were observed in the prepartum fecal VFA or OBCFA profiles among the groups.Prepartum fecal bacterial communities of cows were clustered into two distinct clusters with 70%of the SU cows belonging to cluster 1,in which they represented 60%of the animals.Conclusions:Inter-animal variation in postpartum SARA susceptibility was reflected in post-and prepartum fecal bacterial communities.Differences in prepartum fecal bacterial communities could alert for susceptibility to develop SARA postpartum.Our results generated knowledge on the association between fecal bacteria and SARA development which could be further explored in a prevention strategy.

Coordinated response of milk bacterial and metabolic profiles to subacute ruminal acidosis in lactating dairy cows

Background Bovine milk is an important source of nutrition for human consumption,and its quality is closely associated with the microbiota and metabolites in it.But there is limited knowledge about the milk microbiome and metabolome in cows with subacute ruminal acidosis.Methods Eight ruminally cannulated Holstein cows in mid lactation were selected for a 3-week experiment.The cows were randomly allocated into 2 groups,fed either a conventional diet(CON;40%concentrate;dry matter basis)or a high-concentrate diet(HC;60%concentrate;dry matter basis).Results The results showed that there was a decreased milk fat percentage in the HC group compared to the CON group.The amplicon sequencing results indicated that the alpha diversity indices were not affected by the HC feeding.At the phylum level,the milk bacteria were dominated by Proteobacteria,Actinobacteria,Bacteroidetes,and Firmicutes both in the CON and HC groups.At the genus level,the HC cows displayed an improved proportion of Labrys(P=0.015)compared with the CON cows.Results of both the principal components analysis and partial least squares of discriminant analysis of milk metabolome revealed that samples of the CON and HC groups clustered separately.A total of 31 differential metabolites were identified between the two groups.Of these,the levels of 11 metabolites decreased(α-linolenic acid,prostaglandin E2,L-lactic acid,L-malic acid,3-hydroxysebacic acid,succinyladenosine,guanosine,pyridoxal,L-glutamic acid,hippuric acid,and trigonelline),whereas the levels of the other 20 metabolites increased in the HC group with respect to the CON group(P<0.05).Conclusion These results suggested that subacute ruminal acidosis less impacted the diversity and composition of milk microbiota,but altered the milk metabolic profiles,which led to the decline of the milk quality.

Diarrhea and Acute Renal Injury Culminating in Metformin-Induced Lactic Acidosis

Background: Metformin (M) is an effective first-line hypoglycemic agent in obese type 2 diabetes mellitus due to its low cost and safety profile. The Case: A 66-year-man presented with shock due to lactic acidosis induced by M-supersaturation subsequent to acute renal failure following infective diarrhea. The drug has been used, by this patient, for >10 years without complication. Physical examination, laboratory tests, radiological investigations and blood cultures did not show evidence of new cardiac, hepatic and septic insult. Despite discontinuation of M and 2-days of aggressive hydration, bicarbonate infusions and pressors;toxic levels of the drug persisted and shock-state culminated in severe and oliguric renal failure with serum urea and creatinine up to 50 mmol/L and 1270 umol/L, respectively. Hence, continuous venovenous hemodiafiltration (CVVHDF) was used, for 16-hours, to remove the drug, correct his acidosis and support his severe renal complications. Hours after the procedure;drug level, lactic acidosis and its associated shock improved followed by gradual renal recovery. The patient was discharged after 6 days and serum creatinine reached his base line (180 umol/L) 2 weeks later. The drug was not recommended for his future use. Conclusion: M-induced lactic acidosis, should be considered in assessment of shock in M-treated patients and management of unstable patients indicates early-use of CVVHDF.

Renal calcification in children with renal tubular acidosis:What a paediatrician should know

Renal tubular acidosis(RTA)can lead to renal calcification in children,which can cause various complications and impair renal function.This review provides pediatricians with a comprehensive understanding of the relationship between RTA and renal calcification,highlighting essential aspects for clinical manage-ment.The article analyzed relevant studies to explore the prevalence,risk factors,underlying mechanisms,and clinical implications of renal calcification in children with RTA.Results show that distal RTA(type 1)is particularly associated with nephrocalcinosis,which presents a higher risk of renal calcification.However,there are limitations to the existing literature,including a small number of studies,heterogeneity in methodologies,and potential publication bias.Longitudinal data and control groups are also lacking,which limits our understanding of longterm outcomes and optimal management strategies for children with RTA and renal calcification.Pediatricians play a crucial role in the early diagnosis and management of RTA to mitigate the risk of renal calcification and associated complications.In addition,alkaline therapy remains a cornerstone in the treatment of RTA,aimed at correcting the acid-base imbalance and reducing the formation of kidney stones.Therefore,early diagnosis and appropriate therapeutic interventions are paramount in preventing and managing renal calcification to preserve renal function and improve long-term outcomes for affected children.Further research with larger sample sizes and rigorous methodologies is needed to optimize the clinical approach to renal calcification in the context of RTA in the pediatric population.

A Case of Euglycemic Diabetic Ketoacidosis in Patient with Type 2 DM

Diabetic ketoacidosis (DKA) can cause significant morbidity and mortality in patient with both type 1 and type 2 diabetes mellitus. A subset of DKA cases termed euglycemic diabetic ketoacidosis (EDKA) is characterized by euglycemic (<200 mg/dl), high anion gap metabolic acidosis, and increased plasma ketone concentration. This clinical syndrome is primary related to a general state of starvation, resulting in the development of ketosis while maintaining normoglycemia. It can lead to severe complication, such as extreme dehydration, altered mental status and coma. Early recognition and treatment are essential to avoid this life-threatening complication. EDKA represents approximately 2.6% to 3.2% of total DKA admissions, making it a rare condition. In this case report, a male patient was diagnosed with type 2 DM, 1 week prior to his symptoms and admission in hospital. Despite normal glucose levels at the time of presentation to the ED, he displayed severe acidemia and ketonemia, and was diagnosed with EDKA.

Incomplete distal renal tubular acidosis uncovered during pregnancy:A case report

BACKGROUND Renal tubular acidosis(RTA)is a renal cause of non-anion-gap metabolic acidosis characterized by low urinary ammonia excretion.This condition has a low prevalence,and various congenital and acquired etiologies.To date,only a few cases of idiopathic RTA uncovered during pregnancy have been reported.CASE SUMMARY A previously healthy 32-year-old Korean woman at 30 wk of gestation was admitted to Pusan National University Hospital with preterm labor.At admission,the patient presented with hypokalemia,non-anion-gap metabolic acidosis,and nephrocalcinosis.Distal RTA was diagnosed based on laboratory blood and urine findings and imaging examinations.Various tests,including next-generation gene sequencing panels for nephropathy,were performed to determine the etiology of the disease,which indicated that it was idiopathic.The patient received sodium bicarbonate and potassium chloride supplementation.After 3 wk,she delivered a baby who was subsequently diagnosed with corpus callosum agenesis and colpocephaly.During regular follow-ups for 6 mo postpartum,her hypokalemia and metabolic acidosis were gradually resolved,and medications eventually discontinued.CONCLUSION Herein we describe a case of idiopathic distal RTA discovered during pregnancy.Hypokalemia and metabolic acidosis resolved spontaneously after delivery.

糖尿病乳酸性酸中毒(Diaberic lactic acidosis DLA)的治疗

糖尿病患者可因体内乳酸累积而发生乳酸性酸中毒,如不及时诊断和治疗,可发生死亡。乳酸是葡萄糖的中间代谢产物。葡萄糖的分解代谢包括葡萄糖的有氧氧化和葡萄糖的无氧酵解。前者是葡萄糖在正常有氧条件下彻底氧化产生二氧化碳和水,它是体内糖分解产能的主要途径,大多数组织能获得足够的氧气以供有氧氧化之需而很少进行无氧糖酵解;而后者是葡萄糖在无氧条件下分解成为乳酸,它虽然已非主要代谢途径而且产能有限,但仍是重要的代谢方式并具有病理生理意义。

Intraoperative acidosis is a new predictor for postoperative pancreatic fistula after pancreaticoduodenectomy

BACKGROUND： Early diagnosis of postoperative pancreatic fistula （POPF） is important for proper interventions. The pre- operative, intraoperative and early postoperative biochemical markers have predictive value of POPF. The present study was to evaluate several simple biochemical parameters in the pre- diction of POPF.

Lactic acidosis during telbivudine treatment for HBV: A case report and literature review

All oral nucleoside analogues against hepatitis B virus,with an exception of telbivudine,have been reported causing lactic acidosis(LA).Here we report the first case of chronic hepatitis B developing severe refractory LA during telbivudine monotherapy.A 36-year-old man of Chinese origin received telbivudine antiviral treatment for chronic hepatitis B.After 11 mo of therapy,he developed anorexia,nausea,and vomiting with mild muscle weakness.The patient was found with elevated serum creatine phosphokinase up to 3683 U/L(upper limit of normal 170 U/L)and marked LA.LA did not resolve immediately following discontinuation of telbivudine.His condition began to improve after hemodialysis treatment for 16 times and usage of glucocorticosteroid.The patient fully recovered after 16 wk of treatment.This is the first documented case with severe LA caused by telbivudine monotherapy.Besides serum creatine phosphokinase,blood lactate level should also be closely monitored in patients receiving telbivudine.

Pyruvate is a prospective alkalizer to correct hypoxic lactic acidosis

Type A lactic acidosis resulted from hypoxic mitochondrial dysfunction is an independent predictor of mortality for critically ill patients. However, current therapeutic agents are still in shortage and can even be harmful. This paper reviewed data regarding lactic acidosis treatment and recommended that pyruvate might be a potential alkalizer to correct type A lactic acidosis in future clinical practice. Pyruvate is a key energy metabolic substrate and a pyruvate dehydrogenase(PDH) activator with several unique beneficial biological properties, including anti-oxidant and antiinflammatory effects and the ability to activate the hypoxia-inducible factor-1(HIF-1α)-erythropoietin(EPO) signal pathway. Pyruvate preserves glucose metabolism and cellular energetics better than bicarbonate, lactate, acetate and malate in the efficient correction of hypoxic lactic acidosis and shows few side effects. Therefore, application of pyruvate may be promising and safe as a novel therapeutic strategy in hypoxic lactic acidosis correction accompanied with multi-organ protection in critical care patients.

Changes in feed intake, nutrient digestion, plasma metabolites, and oxidative stress parameters in dairy cows with subacute ruminal acidosis and its regulation with pelleted beet pulp

The objectives of this study were to 1） determine the variation of nutrient digestion, plasma metabolites and oxidative stress parameters triggered by induced subacute ruminal acidosis （SARA）; and 2） evaluate the ability of pelleted beet pulp （BP） as a replacement for ground corn to alleviate SARA. Eight Holstein-Friesian cows were fed four diets during four successive17 day periods： 1） total mixed ration （TMR） containing 0% finely ground wheat （FGW） （WO）; 2） TMR containing 10% FGW （W10）; 3） TMR containing 20% FGW （W20）; and 4） TMR containing 10% BP as a replacement for 10% ground corn （BP10）. The SARA induction protocol reduced the mean ruminal pH from 6.37 to 5.94, and the minimum ruminal pH decreased from 5.99 to 5.41 from baseline to challenge period. Mean ruminal pH increased from 5.94 to 6.05, and minimum daily ruminal pH increased from 5.41 to 5.63, when BP was substituted for corn. The apparent digestibility of nutrients was not affected by the dietary treatments, except that the digestibility of neutral detergent fibre （NDF） and acid detergent fibre （ADF） was reduced in cows fed the W20 diet compared with cows fed the W0 and W10 diets, and cows fed the BP10 diet had higher NDF and ADF digestibility than the cows fed the W20 diet. Cows fed the W20 diet had a lower plasma concentration of 13-hydroxybutyrate （BHBA）, non-esterified fatty acids （NEFA）, cholesterol, triglyceride, and total antioxidative capacity （TAC）, and a higher plasma concentration of glucose, insulin, malonaldehyde （MDA）, super oxygen dehydrogenises （SOD）, and glutathione peroxidase （GSH-Px） than cows fed the W0 diet. Substitution of BP for corn increased concentrations of plasma BHBA and TAC, but decreased concentrations of plasma MDA. Our results indicate that reduction of fibre digestion; the concomitant increase of plasma glucose and insulin; the decrease of plasma BHBA, NEFA, cholesterol, and triglyceride; and changes of plasma oxidative stress parameters are highly related to SARA induced by W20 diets. These variables may be alternative candidates for SARA diagnosis. We also suggest that the substitution of BP for corn could reduce the risk of SARA, increase fibre digestion, and improve the antioxidant status in dairy cows.

Risk factors for mortality in children with diabetic keto acidosis from developing countries

Diabetic keto acidosis(DKA) is the major cause for mortality in children with Diabetes mellitus(DM). With increasing incidence of type 1 DM worldwide, there is an absolute increase of DM among children between 0-14 year age group and overall incidence among less than 30 years remain the same. This shift towards younger age group is more of concern especially in developing countries where mortality in DKA is alarmingly high. Prior to the era of insulin, DKA was associated with 100% mortality and subsequently mortality rates have come down and is now, 0.15%-0.31% in developed countries. However the scenario in developing countries like India, Pakistan, and Bangladesh are very different and mortality is still high in children with DKA. Prospective studies on DKA in children are lacking in developing countries. Literature on DKA related mortality are based on retrospective studies and are very recent from countries like India, Pakistan and Bangladesh. There exists an urgent need to understand the differences between developed and developing countries with respect to mortality rates and factors associated with increased mortality in children with DKA. Higher mortality rates, increased incidence of cerebral edema, sepsis, shock and renal failure have been identified among DKA in children from developing countries.Root cause for all these complications and increased mortality in DKA could be delayed diagnosis in children from developing countries. This necessitates creating awareness among parents, public and physicians by health education to identify symptoms of DM/DKA in children, in order to decrease mortality in DKA. Based on past experience in Parma, Italy it is possible to prevent occurrence of DKA both in new onset DM and in children with established DM, by simple interventions to increase awareness among public and physicians.

Effects of diabetic ketoacidosis in the respiratory system

Diabetes affects approximately 30 million persons in the United States. Diabetes ketoacidosis is one of the most serious and acute complications of diabetes. At the time of presentation and during treatment of diabetic ketoacidosis(DKA), several metabolic and electrolyte derangements can ultimately result in respiratory compromise. Most commonly, hypokalemia, hypomagnesemia and hypophosphatemia can eventually lead to respiratory muscles failure.Furthermore, tachypnea, hyperpnea and more severely, Kussmaul breathing pattern can develop. Also, hydrostatic and non-hydrostatic pulmonary edema can occur secondary to volume shifts into the extracellular space and secondary to increased permeability of the pulmonary capillaries. The presence of respiratory failure in patients with DKA is associated with higher morbidity and mortality. Being familiar with the causes of respiratory compromise in DKA, and how to treat them, may represent better outcomes for patients with DKA.

Use of sodium bicarbonate and blood gas monitoring in diabetic ketoacidosis: A review

Diabetic ketoacidosis(DKA) is a severe and toocommon complication of uncontrolled diabetes mellitus. Acidosis is one of the fundamental disruptions stemming from the disease process, the complications of which are potentially lethal. Hydration and insulin administration have been the cornerstones of DKA therapy; however, adjunctive treatments such as the use of sodium bicarbonate and protocols that include serial monitoring with blood gas analysis have been much more controversial. There is substantial literature available regarding the use of exogenous sodium bicarbonate in mild to moderately severe acidosis; the bulk of the data argue against significant benefit in important clinical outcomes and suggest possible adverse effects with the use of bicarbonate. However, there is scant data to support or refute the role of bicarbonate therapy in very severe acidosis. Arterial blood gas(ABG) assessment is an element of some treatment protocols, including society guidelines, for DKA. We review the evidence supporting these recommendations. In addition, we review the data supporting some less cumbersome tests, including venous blood gas assessment and routine chemistries. It remains unclear that measurement of blood gas pH, via arterial or venous sampling, impacts management of the patient substantially enough to warrant the testing, especially if sodium bicarbonate administration is not being considered. There are special circumstances when serial ABG monitoring and/or sodium bicarbonate infusion are necessary, which we also review. Additional studies are needed to determine the utility of these interventions in patients with severe DKA and pH less than 7.0.

Ketogenic diet versus ketoacidosis： what determines the influence of ketone bodies on neurons？

Glucose is the main energy substrate for neurons, however, at certain conditions, e.g. in starvation, these cells could also use ketone bodies. This approach is used in clinical conditions as the ketogenic diet. The ketogenic diet is actually a biochemical model of fasting. It includes replacing carbohydrates by fats in daily meal. Synthesis of ketone bodies β-hydroxubutirate, acetoacetate and acetone begins once glycogen stores have depleted in the liver. The ketogenic diet can be used to treat clinical conditions, primarily epilepsy. The mechanism of neuroprotective action of ketogenic diet is not very clear. It is shown that ketone bodies influence neurons at three different levels, namely, metabolic, signaling and epigenetic levels. Ketone bodies are not always neuroprotective. Sometimes they can be toxic for the brain. Ketoacidosis which is a very dangerous complication of diabetes mellitus or alcoholism can be taken as an example. The exact mechanism of how neuroprotective properties of ketone bodies reverse to neurotoxic is yet to be established.

The Effect of Hypercapnic Acidosis Preconditioning on Rabbit Myocardium

This study observed the protective effect of hypercapnic acidosis preconditioning on rabbit heart suffered from ischemia-reperfusion injury. Hypercapnic acidosis was established in animals with mechanical hypoventilation before ischemia-reperfusion. Thirty-two rabbits were randomly divided into 4 groups, with each having 8 aminals in term of the degree of acidification： hypercapnic acidosis group A （group A）, hypercapnic acidosis group B （group B）, hypercapnic acidosis group C （group C）, ischemia and reperfusion group （group IR）. Animals in group IR were ventilated normally （tidal volume： 15 mL/kg, breathing rate 35 bpm）. The PETCO2 was maintained at the level of 40-50 mmHg for 30 min. Animals in groups A, B, C received low-frequency, low-volume ventilation to achieve hypercarbonic acidosis and the target levels of PETCO2 were 75-85 ,65-75, 55-65 mmHg, respectively, with levels being maintained for 5 min. The animals then were ventilated normally to lower PETCO2 to 40-50 mmHg. The left anterior branch artery of all the animals was ligated for 30 min and reperfused for 180 min. Then the infarct size was calculated. The cardiomyocytes were morphologically observed and ECG and hemodynamics were monitored on continuous basis. Acid-base balance was measured during procedure. Our results showed that the infarct size was （48.5±11.5）% of the risk area in the control group and （42.4±7.9）% in group C （P〉0.05）. Mean infarct size was significantly smaller in group B （34.5%±9.4%） （P〈0.05 vs control group） and group A （31.0%±9.1%） （P〈0.01 vs control group）. It is concluded that HA-preconditioning can effectively protect the myocardium.

EVALUATION OF MITOCHONDRIAL ENCEPHALOMYOPATHY WITH LACTIC ACIDOSIS AND STROKE-LIKE EPISODES WITH MAGNETIC RESONANCE IMAGING AND PROTON MAGNETIC RESONANCE SPECTROSCOPY

Objective To study the characteristics of spectra on proton magnetic resonance spectroscopy （^1H-MRS） and its value in patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes （MELAS）. Methods Seven clinically diagnosed patients with MELAS underwent magnetic resonance imaging （MRI） and ^1H-MRS examinations. The ^1H-MRS techniques, characteristics of the spectra, and its correlation with the laboratory tests were analyzed. Reaults Cerebral abnormalities were revealed in all 7 patients on conventional MR images, and most abnormal signals were observed in bilateral occipital, parietal, and temporal lobes. We found 4 cases with basal ganglia involvement, 2 cases with mild frontal lobe lesions, and 1 case with involvement of lateral cerebral peduncles and thalami. Additionally, 1 patient was involved with left insular lobe. Spectra from prominent lesions in brain parenchyma showed lactate doublet peak in 6 patients, 3 of whom were also noted lactate peak in ventricular cerehrospinal fluid （CSF）. Conclusion ^1H-MRS may provide more direct information about the metabolism changes, which aids to affirm the diagnosis, and may replace the conventional invasive method of quantifying lactate in CSF.

Euglycemic diabetic ketoacidosis:A missed diagnosis

Euglycemic diabetic ketoacidosis(DKA)is an acute life-threatening metabolic emergency characterized by ketoacidosis and relatively lower blood glucose(less than 11 mmol/L).The absence of hyperglycemia is a conundrum for physicians in the emergency department and intensive care units;it may delay diagnosis and treatment causing worse outcomes.Euglycemic DKA is an uncommon diagnosis but can occur in patients with type 1 or type 2 diabetes mellitus.With the addition of sodium/glucose cotransporter-2 inhibitors in diabetes mellitus management,euglycemic DKA incidence has increased.The other causes of euglycemic DKA include pregnancy,fasting,bariatric surgery,gastroparesis,insulin pump failure,cocaine intoxication,chronic liver disease and glycogen storage disease.The pathophysiology of euglycemic DKA involves a relative or absolute carbohydrate deficit,milder degree of insulin deficiency or resistance and increased glucagon/insulin ratio.Euglycemic DKA is a diagnosis of exclusion and should be considered in the differential diagnosis of a sick patient with a history of diabetes mellitus despite lower blood glucose or absent urine ketones.The diagnostic workup includes arterial blood gas for metabolic acidosis,serum ketones and exclusion of other causes of high anion gap metabolic acidosis.Euglycemic DKA treatment is on the same principles as for DKA with correction of dehydration,electrolytes deficit and insulin replacement.The dextrosecontaining fluids should accompany intravenous insulin to correct metabolic acidosis,ketonemia and to avoid hypoglycemia.

Mucosal necrosis of the small intestine in myopathy,encephalopathy,lactic acidosis,and stroke-like episodes syndrome

This report presents a case of massive mucosal necrosis of the small intestine in a patient with mitochondrial myopathy,encephalopathy,lactic acidosis,and stroke-like episodes(MELAS),which particularly affects the brain,nervous system and muscles.A 45-year-old Japanese female,with an established diagnosis of MELAS,presented with vomiting.Computed tomography showed portomesenteric venous gas and pneumatosis intestinalis.She underwent a resection of the small intestine.A microscopic study showed necrosis of the mucosa and vacuolar degeneration of smooth muscle cells in the arterial wall.Immunohistochemistry showed anti-mitochondrial antibody to be highly expressed in the crypts adjacent the necrotic mucosa.The microscopic and immunohistochemical findings suggested the presence of a large number of abnormal mitochondria in MELAS to be closely linked to mucosal necrosis of the small intestine.