Comparative Metabolic Analysis of Lactate for CHO Cells in Glucose and Galactose

Camila A. Wilkens,Claudia Altamirano,Ziomara P. Gerdtzen 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.4

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so,Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H+ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.

Lactate Can Modulate the Expression of Lactate Dehydrogenase and Aquaporin Genes in Mouse Preimplanation Embryos

Soojung Shin,전용필 한국발생생물학회 2012 발생과 생식 Vol.16 No.3

It is suggested that carbohydrate metabolites may involve in the development of morula to blastocyst but many of the mechanisms are not unmasked. Two-cell stage embryos were collected and examined the effects of lactate on the development of blastocyst in vitro. The expression profiles of lactate dehydrognase (Ldh) genes and aquaporin (Aqp)genes were analyzed with RT-PCR. The successful development from morula to blastocyst was dependent on lactate concentrations. The expression profiles of Ldh genes were changed by the lactate concentration. Ldha was expressed in morula stage at 10 mM lactate, and in blastocyst stage at lactate free condition. Ldhb was expressed in morula stage at 10 mM and 20 mM lactate, and in blastocyst stage at 10 mM lactate. Aqp genes were also showed different expression patterns by the lactate concentrations. Aqp3 was expressed in hatching embryo at 120 hr post hCG administration (hph)which was cultured in BWW medium and lactate free condition. Aqp7 was expressed in hatching embryos at 120 hph which was cultured at 10 mM lactate condition. Also Aqp8 was expressed in hatching embryo at BWW and 20 mM lactate condition. Aqp9 was expressed in morula at BWW and 10 mM lactate condition, and in blastocyst at BWW. Based on these results, it is suggested that concentration of lactate in the medium and the level of lactate synthesis in embryo is critical factor for blastocoels formation. In addition it is suggested that LDH may involve the AQPs expression in embryos.

Lactate Can Modulate the Expression of Lactate Dehydrogenase and Aquaporin Genes in Mouse Preimplanation Embryos

Shin Soo-Jung,Cheon Yong-Pil 한국발생생물학회 2012 발생과 생식 Vol.16 No.3

It is suggested that carbohydrate metabolites may involve in the development of morula to blastocyst but many of the mechanisms are not unmasked. Two-cell stage embryos were collected and examined the effects of lactate on the development of blastocyst in vitro. The expression profiles of lactate dehydrognase (Ldh) genes and aquaporin (Aqp) genes were analyzed with RT-PCR. The successful development from morula to blastocyst was dependent on lactate concentrations. The expression profiles of Ldh genes were changed by the lactate concentration. Ldha was expressed in morula stage at 10 mM lactate, and in blastocyst stage at lactate free condition. Ldhb was expressed in morula stage at 10 mM and 20 mM lactate, and in blastocyst stage at 10 mM lactate. Aqp genes were also showed different expression patterns by the lactate concentrations. Aqp3 was expressed in hatching embryo at 120 hr post hCG administration (hph) which was cultured in BWW medium and lactate free condition. Aqp7 was expressed in hatching embryos at 120 hph which was cultured at 10 mM lactate condition. Also Aqp8 was expressed in hatching embryo at BWW and 20 mM lactate condition. Aqp9 was expressed in morula at BWW and 10 mM lactate condition, and in blastocyst at BWW. Based on these results, it is suggested that concentration of lactate in the medium and the level of lactate synthesis in embryo is critical factor for blastocoels formation. In addition it is suggested that LDH may involve the AQPs expression in embryos

Association of lactate clearance with outcomes of patients with gastrointestinal bleeding visiting the emergency department

고해원,오재훈,강형구,임태호,고벽성 대한응급의학회 2022 대한응급의학회지 Vol.33 No.4

Objective: Lactate clearance is reportedly associated with the outcomes of various critical illnesses. However, few studies have examined the association between lactate clearance and outcomes in patients with gastrointestinal bleeding (GIB). Methods: A single-center retrospective observational study between 2016 and 2020 was conducted. Consecutive adult patients with GIB symptoms (melena, hematemesis, and hematochezia) presenting to the emergency department were included. Lactate clearance was calculated as ([initial lactate-subsequent lactate]/initial lactate)×100. The association between lactate clearance and outcomes was examined by multivariable logistic regression analysis. The primary outcome was the in-hospital mortality. The area under the curve (AUC) of lactate clearance for in-hospital mortality was calculated. The sensitivity and specificity with optimal cutoff values were computed. The AUC of lactate clearance was compared with the Glasgow-Blatchford score and AIMS65 for predicting in-hospital mortality. Results: Three hundred and fifty-one patients were included in the final analysis, and the in-hospital mortality rate was 12%. Lactate clearance was significantly associated with lower in-hospital mortality (odds ratio, 0.991; P=0.016). The AUC of lactate clearance for in-hospital mortality was 0.64. The AUC of Glasgow-Blatchford score and AIMS65 for inhospital mortality was not significantly different from lactate clearance (P=0.759 and P=0.442, respectively). A cutoff lactate clearance of less than 10% had 45.2% sensitivity, 30.7% specificity, 8.2% positive predictive value, and 80.5% negative predictive value for predicting in-hospital mortality. Conclusion: Lactate clearance was independently associated with in-hospital mortality in GIB patients. Further prospective studies will be needed to address the prognostic value of lactate clearance in GIB.

Higher Lactate Level and Lactate-to-Pyruvate Ratio in Autism Spectrum Disorder

오미애,김순애,유희정 한국뇌신경과학회 2020 Experimental Neurobiology Vol.29 No.4

Mitochondrial dysfunction is considered one of the pathophysiological mechanisms of autism spectrum disorder (ASD). However, previous stud- ies of biomarkers associated with mitochondrial dysfunction in ASD have revealed inconsistent results. The objective of this study was to evaluate biochemical markers associated with mitochondrial dysfunction in subjects with ASD and their unaffected family members. Lactate and pyruvate levels, as well as the lactate-to-pyruvate ratio, were examined in the peripheral blood of probands with ASD (Affected Group, AG) and their unaf- fected family members (biological parents and unaffected siblings, Unaffected Group, UG). Lactate ≥22 mg/dl, pyruvate ≥1.4 mg/dl, and lactate-to- pyruvate ratio >25 were defined as abnormal. The clinical variables were compared between subjects with higher (>25) and lower (≤25) lactate-to- pyruvate ratios within the AG. The AG (n=59) had a significantly higher lactate and lactate-to-pyruvate ratio than the UG (n=136). The frequency of subjects with abnormally high lactate levels and lactate-to-pyruvate ratio was significantly higher in the AG (lactate 31.0% vs. 9.5%, ratio 25.9% vs. 7.3%, p<0.01). The relationship between lactate level and the repetitive behavior domain of the Autism Diagnostic Interview-Revised was statis- tically significant. These results suggest that biochemical markers related to mitochondrial dysfunction, especially higher lactate levels and lactate- to-pyruvate ratio, might be associated with the pathophysiology of ASD. Further larger studies using unrelated individuals are needed to control for the possible effects of age and sex on chemical biomarker levels.

The Effectiveness of Training Routine with Reference to the Physiological Demand of Squash Match Play

( Pralay Majumdar ),( Malay Mandal ),( Devashish Yadav ) 한국스포츠정책과학원(구 한국스포츠개발원) 2009 International Journal of Applied Sports Sciences Vol.21 No.1

Purpose: The present study was taken up to monitor the physiological demands of squash play based on heart rate and lactate response while in a competitive situation. Four on court specific training programs were evaluated based on lactate response and performance time to find out their effectiveness with the actual demands of the game. Method: 33 male and 9 female junior squash players volunteered for this study. Body fat was calculated indirectly from skin fold thickness a using Harpenden skin fold caliper at four sites: biceps, triceps, sub scapular and supra iliac. Body fat was calculated from body density. The cardio respiratory variables (VO2 max) at maximal effort were determined during a graded protocol of running on a treadmill starting from 8 Km/hr and increasing every 2 min. at the rate of 2 km/hr till volitional exhaustion. The expired air sample was analyzed by Gas Analyzer (Oxycon Champion-Germany). Four on court drills were monitored and lactate level and timing/court coverage were registered. For estimating whole blood lactate, capillary blood samples were drawn from a finger tip between 2 min and 3 min. after the cessation of the activity. The samples were analyzed in a calibrated automatic lactate analyzer in order to estimate whole blood lactate. The data were subjected to statistical treatment such as mean and standard deviation. One-way ANOVA were computed to assess the difference in heart rate and lactate among the three games. Result: Repeated measures of ANOVA demonstrated no significance difference observed in gender in terms of heart rate and lactate response during three games. The heart rate response in males ranged from 157-177, 162-182 and 165-182 beats. min-1 for games 1, 2 and 3 respectively. In females, the heart rate response in 3 games revealed 156-181, 165-183 and 163-181 beats. min-1. Peak game lactate found for males and females were 4.10 and 4.14 mM.l-1 achieved after the third and second game respectively. Estimated peak lactate in four on court drills i.e. ghosting, court run, agility and box sprints were 10.66±3.07, 14.00±1.79, 16.90±2.80 and 13.11±2.40 for males and 12.251.79, 13.78±2.17, 17.30±2.53 and 13.46±4.37 mM.l-1 for females respectively. Conclusion: In all the on court drills, the level of blood lactate was much higher than game lactate. The higher lactate levels cause metabolic acidosis and therefore may not prove to be an effective training routine unless modified in terms of duration/intensity to optimize training benefit.

액체크로마토그래프-탠덤질량분석기(LC-MS/MS)를 이용한 소변 내 D-, L- Lactate 분리 및 정량

문철진,양송현,Moon, Chul Jin,Yang, Song Hyun 대한유전성대사질환학회 2015 대한유전성대사질환학회지 Vol.15 No.2

사람의 신체내에서 주로 존재하는 lactate는 L-lactate로서 몇몇 선천성대사이상과 관련하여 증가된다. 최근 2형 당뇨병(type 2 diabetes)과 만성적인 염증성 위장질환(inflammatory bowel disease)과 관련하여 증가되는 D-lactate를 L-lactate와 분리해야 하는 요구도가 높아졌다. 이에 액체크로마토그래프-탠덤질량 분석기를 사용하여 소변 내 D-, L-lactate를 분리하는 방법을 확립하였으며, 분석시간과 정밀성, 정확성, 특이성 등에서 신뢰성 있는 방법임을 확인하였다. Purpose: Lactate has two optical isomers, L-lactate and D-lactate. In human L-lactate is the most abundant enantiomer of lactate. As plasma and urinary levels of L-lactate is associated with inherited metabolic disorders in general, D-lactate have been linked to the presence of diabetes and inflammatory bowel disease. Previously developed techniques have shown several limitations to further evaluate D-lactate as a biomarker for this condition. In this paper, we describe a highly sensitive, specific and fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of D-, L-lactate in urine. Methods: D- and L-lactate were quantified using high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) with labelled internal standard. Samples were derivatized with (+)-O,O'-diacety-L-tartaric anhydride (DATAN) and seperated on a Poroshell 120 EC-C18 column. Results: Quantitative analysis of D-, and L-lactate was achieved successfully. Calibration curves were linear (r>0.999) over $0.5-100{\mu}g/mL$. Stabilities for samples were within the 10% varation. Inter- and Intra-day assay variations were below 10%. Conclusion: The presented method proved to be suitable for the quantitation of D- and L-lactate and opens the possibility to explore the use of D-lactate as a biomarker.

Genome-Wide Transcriptomic Analysis of n-Caproic Acid Production in Ruminococcaceae Bacterium CPB6 with Lactate Supplementation

( Shaowen Lu ),( Hong Jin ),( Yi Wang ),( Yong Tao ) 한국미생물 · 생명공학회 2021 Journal of microbiology and biotechnology Vol.31 No.11

n-Caproic acid (CA) is gaining increased attention due to its high value as a chemical feedstock. Ruminococcaceae bacterium strain CPB6 is an anaerobic mesophilic bacterium that is highly prolific in its ability to perform chain elongation of lactate to CA. However, little is known about the genome-wide transcriptional analysis of strain CPB6 for CA production triggered by the supplementation of exogenous lactate. In this study, cultivation of strain CPB6 was carried out in the absence and presence of lactate. Transcriptional profiles were analyzed using RNA-seq, and differentially expressed genes (DEGs) between the lactate-supplemented cells and control cells without lactate were analyzed. The results showed that lactate supplementation led to earlier CA p,roduction, and higher final CA titer and productivity. 295 genes were substrate and/or growth dependent, and these genes cover crucial functional categories. Specifically, 5 genes responsible for the reverse β-oxidation pathway, 11 genes encoding ATP-binding cassette (ABC) transporters, 6 genes encoding substrate-binding protein (SBP), and 4 genes encoding phosphotransferase system (PTS) transporters were strikingly upregulated in response to the addition of lactate. These genes would be candidates for future studies aiming at understanding the regulatory mechanism of lactate conversion into CA, as well as for the improvement of CA production in strain CPB6. The findings presented herein reveal unique insights into the biomolecular effect of lactate on CA production at the transcriptional level.

Enhancement of Erythropoietin Production in Recombinant Chinese Hamster Ovary Cells by Sodium Lactate Addition

Choi, Yeon-Sook,Lee, Doo-Young,Kim, Ick-Young,Kim, Hong-Jin,Park, Hong-Woo,Choe, Tae-Boo,Kim, Ik-Hwan Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.1

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However, the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.

Inclusion of lactate level measured upon emergency room arrival in trauma outcome prediction models improves mortality prediction: a retrospective, single-center study

문종환,황경진,윤덕용,정경원 대한중환자의학회 2020 Acute and Critical Care Vol.35 No.2

Background: This study aimed to develop a model for predicting trauma outcomes by adding arterial lactate levels measured upon emergency room (ER) arrival to existing trauma injury severity scoring systems. Methods: We examined blunt trauma cases that were admitted to our hospital during 2010– 2014. Eligibility criteria were cases with an Injury Severity Score of ≥9, complete Trauma and Injury Severity Score (TRISS) variable data, and lactate levels that were assessed upon ER arrival. Survivor and non-survivor groups were compared and lactate-based prediction models were generated using logistic regression. We compared the predictive performances of traditional prediction models (Revised Trauma Score [RTS] and TRISS) and lactate-based models using the area under the curve (AUC) of receiver operating characteristic curves. Results: We included 829 patients, and the in-hospital mortality rate among these patients was 21.6%. The model that used lactate levels and age provided a significantly better AUC value than the RTS model. The model with lactate added to the TRISS variables provided the highest Youden J statistic, with 86.0% sensitivity and 70.8% specificity at a cutoff value of 0.15, as well as the highest predictive value, with a significantly higher AUC than the TRISS. Conclusions: These findings indicate that lactate testing upon ER arrival may help supplement or replace traditional physiological parameters to predict mortality outcomes among Korean trauma patients. Adding lactate levels also appears to improve the predictive abilities of existing trauma outcome prediction models.