谷氨酰胺酶Gls1最新综述

Review Article

Key Roles of Glutamine Pathways in Reprogramming

the Cancer Metabolism

Krzysztof Piotr Michalak,1,2Agnieszka Ma T kowska-K w dziora,3

Bogus B aw Sobolewski,4and Piotr Wo f niak4

1Laboratory of Vision Science and Optometry,Faculty of Physics,Adam Mickiewicz University of Pozna′n,

Umultowska Street85,61-614Pozna′n,Poland

2Nanobiomedical Center of Pozna′n,Umultowska Street85,61-614Pozna′n,Poland

3Department of Clinical Pharmacology,Chair of Cardiology,Pozna′n University of Medical Sciences,D?uga Street1/2,

61-848Pozna′n,Poland

4Polish Mother’s Memorial Hospital-Research Institute,Outpatient Clinic,Rzgowska Street281/289,?′o d′z,Poland

Correspondence should be addressed to Krzysztof Piotr Michalak;kmichalak@https://www.wendangku.net/doc/469836091.html,.pl

Received20March2015;Revised7April2015;Accepted8April2015

Academic Editor:Claudio Cabello-Verrugio

Copyright?2015Krzysztof Piotr Michalak et al.This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original work is properly cited.

Glutamine(GLN)is commonly known as an important metabolite used for the growth of cancer cells but the effects of its intake in cancer patients are still not clear.However,GLN is the main substrate for DNA and fatty acid synthesis.On the other hand,it reduces the oxidative stress by glutathione synthesis stimulation,stops the process of cancer cachexia,and nourishes the immunological system and the intestine epithelium,as well.The current paper deals with possible positive effects of GLN supplementation and conditions that should be fulfilled to obtain these effects.The analysis of GLN metabolism suggests that the separation of GLN and carbohydrates in the diet can minimize simultaneous supply of ATP(from glucose)and NADPH2(from glutamine)to cancer cells.It should support to a larger extent the organism to fight against the cancer rather than the cancer cells.GLN cannot be considered the effective source of ATP for cancers with the impaired oxidative phosphorylation and pyruvate dehydrogenase inhibition.GLN intake restores decreased levels of glutathione in the case of chemotherapy and radiotherapy;thus,it facilitates regeneration processes of the intestine epithelium and immunological system.

1.Introduction

The development of cancer therapy is the urgent aim for science today.Growing knowledge about the metabolism of cancer cells provides new interesting hints concerning the metabolic targeting of the treatment and searching for new drugs inhibiting the growth of cancer[1].The current paper presents the review of recently developed biochemical aspects of cancer metabolism and possible use of this knowledge for targeting the therapy into mainstream metabolic enzymes. Main emphasis is placed on the possible effect of glutamine (GLN)supplementation as a nutrient supporting both the cancer growth and organism to fight against the cancer.The individual kinds of tumors are characterized by different metabolic alterations which determine possible positive or negative effect of GLN supplementation.In the current paper, different aspects of cancer metabolism are discussed and analyzed in this context.

The aim of this paper is to point especially to these aspects of GLN metabolism that could be positively used while planning the treatment of the cancer patient and to point to conditions that should be fulfilled in order to make the positive effects of GLN supplementation surpass the negative ones.

2.Metabolism of Glutamine

2.1.Metabolism of Glutamine in Healthy Cells.GLN is one of20amino acids,commonly existing in every protein.The

Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2015, Article ID 964321, 14 pages https://www.wendangku.net/doc/469836091.html,/10.1155/2015/964321

mammal organism is able to synthesize it.GLN is a central

point in the metabolism of majority of amino acids[2].

The first stage of amino acids metabolism consists in

its change inside muscles tissue into the GLN and to lower

degree into the alanine.The amino acids are metabolized

to different tricarboxylic acid(TCA)cycle metabolites,enter

TCA cycle,and go away asα-ketoglutarate(αKT)or alanine.αKT is next metabolized to GLN(Figure1).Then,GLN is carried to other tissues like hepar,immunological system,

intestinal tract,and fibroblasts where it is used up as the

priority fuel[3,4].For this reason GLN comprises60%of

the total free amino acid pool in the blood plasma[5].GLN

synthesis rapidly decreases in the deficiency of energy in the

cell.It occurs due to the fact that the energy from ATP and

NADH2has to be supplied to the synthesis of glutamine

(Figure1).

First,in Figure1let us analyze two reactions that metabo-

lize reciprocal reactions between GLN and glutamate(GLU).

These reactions are catalyzed by two different enzymes.It

results from the relatively high free Gibbs energy between

both substances.The decay of GLN occurs easily and with

relatively high loss of free energy.In the opposite,the ATP

is necessary to synthesize GLN.Thus,in the case of low ATP

level in the cell,the equilibrium of this reaction is strongly

shifted to the left(GLU creation).

The relation betweenαKT and GLU is more complicated.

Three enzymes are capable of catalyzing this reaction[4]. Transaminase(TA19)carries the amine group from other amino acids into theαKT and it makes it possible to transfer the nitrogen into the urine cycle.The l-amino acid oxidase (AAO20)is the enzyme that acts only in one direction.It catalyses the deamination of GLU and produces the ammonia for urine cycle.The hydrogen peroxide that is generated in this reaction is quickly decayed by catalase and it makes this reaction irreversible.This reaction is characterized by the sig-nificant loss of free energy,as well.The third,crucial enzyme is the glutamate dehydrogenase(GDH21).This reaction is characterized with K eq≈1.It means that the direction of the reaction depends on the metabolic state of the organism. It should be noted that the decay of GLU is combined with production of NADH2or NADPH2.Both these molecules are high energy compounds and its free energy corresponds approximately to the energy of3ATP molecules.The activity of GDH21is regulated by allosteric inhibitors,ATP,NADH2 and by the activator,ADP,as well.Thus,this enzyme is active in the state of the lack of energy in the cell.It makes the biological sense.In the case of high energy level in the cell(high concentration of ATP,NAD(P)H2,andαKT),the synthesis of GLU should dominate.This would open the minicycle between GLU andαKT catalyzed by GDH21and AAO20and would lead to the dissipation of energy stored in NAD(P)H2.Thus,GDH21is turned off.In the case of insufficient energy level in the cell the quick energy pathway from GLN and GLU is open.The result is the increase of GLU and GLN decay and the decrease in GLN levels in the blood. It causes the activation of muscle decay in order to maintain the GLN levels in the blood and provide the GLN to the cells that need it.The tissues that especially need the energy for the fight against the tumor and for repairing damages after chemo-and radiotherapy are the intestine epithelium and the immune system[6–21].

The gluconeogenesis is the second reason of quicker GLN metabolism in cancer patients,especially in the advanced state of the illness.It is known that cancers use the glucose as the main source of ATP,according to the so-called Warburg effect[22].In the case of high glucose intake by the cancer,the glucose is restored in the liver from amino acids and GLN is the main source of amino acids in this process[23].

Thus,GLN is released from muscles in the periods of increased metabolic stress and the concentration of the intracellular GLN decreases by more than50%[24,25].The resynthesis of GLN cannot be sustained on the exact level in these periods.

The suggested glutamine requirement after uncompli-cated major operations,major injury,gastrointestinal mal-functions,and during cachexia is～0.15–0.20g glutamine per kg body weight.In patients with serious immune deficiency, after bone marrow transplantation,during episodes of sepsis, systemic inflammatory response syndrome,or multiorgan failure,the requirement is increased to～0.3–0.5g glutamine per kg body weight[5,26–28].

It is difficult to provide this amount of GLN to the diet because generally the intake of natural GLN does not exceed 10g.Thus,although the organism is capable of synthesizing the GLN,it should be treated as deficient nutrient in the periods of increased metabolic stress.

2.2.Metabolism of Glutamine in Cancer Cells.The cancer cells differ significantly from the normal cells in the intensity of the main metabolic pathways.The most important differences consist in the following:

(i)higher level of the oxidative stress accompanying the

metabolic malfunctions[25,29–36],

(ii)increased aerobic glycolysis and production of lactic acid(Warburg effect)[22],

(iii)production of ATP mainly in the aerobic glycolysis process[22,34,37,38],

(iv)reduction of the activity of pyruvate dehydrogenase complex PDHC6that converts pyruvate to mitochon-

drial Acetyl-CoA[34,37,39](the upper indices at the

enzyme abbreviation point to the reaction numbers in

Figures1and2),

(v)reduction of intensity of TCA cycle due to reduced activity of some TCA cycle enzymes[29–31,33,40,

41],

(vi)reduction of activity of the oxidative phosphorylation (OXPH)in the cytochrome chain[25,29–31,33], (vii)reduced activity of the pentose phosphate cycle and production of NADPH2for fatty and nucleic acid

synthesis mainly by the malic enzyme(ME16), (viii)high utilization of glutamine mainly for the pro-duction of NAPDH2that is used mainly for fatty

and nucleic acid synthesis and for restoration of the

GTP

NADH

Figure1:The connection between glutamine and tricarboxylic acid cycle:glutamine is metabolized to glutamate and next toα-ketoglutarate. Quick energy pathway from glutamine is open if glutamate dehydrogenase is activated by the low ATP and high ADP levels(as in majority of cancers).In the opposite,ATP and NAD(P)H2are necessary for the synthesis of glutamine which takes place especially in muscles.Reactions 20,21,and22are the source of ammonia and ammonia induced autophagy.(Indices at enzyme abbreviations point to reaction numbers in Figures1and2.)

molecules of TCA cycle that leave mitochondria for

different anabolic purposes of the intensively dividing

cells[42,43],

(ix)high activity of ATP citrate lyase(ACL18)in the cancer cells that produce cytoplasmatic Acetyl-CoA

due to higher demand of the cancer cells for the fatty

acid synthesis[44,45],

(x)increased activity of hypoxia inducible factor(HIF-1α)in cancer cells both in the hypoxic[46–48]and

normoxic conditions[49].

Glutamine is the controversial diet supplement that can modify the metabolism in both cancer and normal cells. Different aspects of GLN point either to the potential dis-advantages or to potential benefits of its supplementation in cancer patients.GLN is recently presented to be one of the main nutrients for cancer growth[42,50].The most important role consists in supplying the reduced hydrogen in the form of NADPH2.NADPH2is next utilized by cancer cells mainly for the fatty and nucleic acids synthesis.GLN can be also the significant source for gluconeogenesis in the liver, being the source of glucose for the cancer.

On the opposite,many papers point to the positive role of GLN supplementation.Let us summarize the potential beneficial effects of GLN supplementation[23]:

(1)stimulation of NK lymphocytes metabolism and

intestine mucosa regeneration[12,51–54];

(2)reduction of the side effects of chemo-and radiother-

apy and especially the reduction of intestinal mucosa

injures[13–21,55];

(3)increase in some therapeutic effect of chemotherapy,

among other things by the increased concentration of

some drugs inside the tumor cells[15,18].

In the next step,let us deal with these aspects that exhibit the potential slowing down effect on tumor growth,however,accompanied by increase in the resistance to starvation and/or oxidative stress:

(4)activation of the autophagy in the cancer cells due

to the increased ammonia production.Autophagy is

the process of self-digestion which makes it possi-

ble to recycle the cellular proteins and lipids into

its metabolic precursors.This process promotes cell

survival in the case of starvation or other metabolic

stresses[42,56];

(5)stimulation of the glutathione synthesis which

inhibits the oxidative stress in healthy cells and

contributes to the cancer cell growth inhibition

[57–59].It can make,however,the cancer cells more

resistant to chemo-and radiotherapy[60].

In analyzing GLN intake as a possible positive or negative factor supporting the cancer growth and/or cancer treatment, one must take into consideration the differences between the metabolism of healthy and cancer cells[2].The main problem of this analysis is the variety of metabolic changes characterizing different cancer types.Individual types of cancer metabolism should be analyzed with regard to the possible positive or negative effect of glutamine supplemen-tation.Some methods of metabolic analysis of cancer cells are available[39,61,62].

At first,the division into two main groups of cancer types should be analyzed:cancers with the(a)normal and (b)reduced activity of pyruvate dehydrogenase complex (PDHC6).The activity of this enzyme is deteriorated in the majority of cancers[34,37,39].Theoretical analysis of GLN degradation presented in this paper shows that the GLN supplementation may be beneficial especially in cancers with reduced PDHC6activity.

Figure2:General scheme of glutamine(GLN)utilization in main metabolic pathways with details concerning ATP,NADH2,and NADPH2 production/utilization.PDHC6catalyzing the reaction pyruvate→Acetyl-CoA is the only reaction that is able to reduce the total number of TCA molecules in the cell;thus,every GLN degradation pathway goes to pyruvate.Two pathways can be defined(see also Figure3): (1)glutaminolysis(GLL):GLN→αKT→SC→malate→(to cytoplasm)→pyruvate and(2)reverse-TCA(R-TCA):GLN→citrate→(to cytoplasm)→oxaloacetate→malate→pyruvate.The balance of GLL pathway is+1FADH2,+1NADPH2,and+1ATP(alternatively0ATP in the case of reaction12).The balance of R-TCA pathway is?2NADH2,?1ATP,and+1NADPH2.The alternative NAD(P)H2from the reaction 21is omitted.The conversion of Acetyl-CoA to malonyl-CoA in the fatty acid synthesis utilizes1additional ATP.Only one ATP molecule can be created directly during degradation of GLN in the GLL pathway(reaction11).R-TCA utilizes rather ATP(for enzyme names,see enzyme abbreviation list).

3.Glutamine and Glutathione

Glutamine is significantly involved in the synthesis of glu-tathione(GSH)—the tripeptide that comprises three amino acids:glutamic acid,cysteine and glycine.This compound serves as a very important intracellular antioxidant and detoxication factor.Besides working as a scavenger of reactive oxygen species(ROS),GSH is involved in a variety of other metabolic functions such as DNA repair,activation of transcription factors,cell cycle regulation,modulation of calcium homeostasis,and regulation of enzyme activity.Most of these functions of GSH are related to its ability to maintain

Reverse TCA (R-TCA)

Acetyl-CoA pathway

Figure3:The schematic presentation of two glutamine metabolism pathways:glutaminolysis and reverse TCA and its connection with the Acetyl-CoA pump that transports Acetyl-CoA from mitochondria to cytoplasm.In the case of deteriorated PDHC6activity as in majority of cancers,the carbon skeleton of GLN can be metabolized only to lactate or alanine.The other pathways are rather negligible.

reduced cellular environment[60,63].Malignant diseases are accompanied by GLN deficiency and reduction of GSH in the host organism,which can be reversed by dietary GLN[58].In addition,several reports suggested that the GSH constituent amino acids,including GLN,inhibit tumor promotion,at least in part,by their interference with GSH metabolism [57,59].

The benefits of glutamine supplementation in cancers through the influence on GSH metabolism were broadly presented by Todorova et al.[60].Tumor cells are shown to have higher concentration of reduced(active)form of GSH than the surrounding normal cells,which contributes to higher rate of cell proliferation and resistance to chemo-and radiotherapy.Therefore,selective tumor depletion of GSH presents a promising strategy in cancer treatment.Todorova et al.[60]have examined the effects of GLN on GSH levels in7,12-dimethylbenz[α]anthracene-(DMBA-)induced mammary tumors and correlated the results with protein and mRNA expression of apoptosis-related proteins Bcl-2,Bax, and caspase-3in tumor cells.The results have shown that GLN supplementation caused a significant decrease by57% in tumor GSH levels and similar ratio GSH/oxidized GSH (GSSG)accompanied by upregulation of Bax and caspase-3(apoptosis induced factors)and downregulation of Bcl-2

(apoptosis inhibiting factor).Bcl-2is known to play a role in promoting cell survival and inhibition of apoptosis,while Bax,a member of the Bcl-2family can induce apoptosis. Caspase-3is the main apoptosis-induced enzyme.In the GLN supplemented group Bax mRNA has increased by19%and caspase-3mRNA by30%and Bcl-2mRNA decreased by33%.

The importance of GSH depletion and reduction of GSH/GSSG ratio for stimulation of apoptosis has also been demonstrated in several in vitro models[59,64–66].GSH depletion was found to be necessary and sufficient to induce cytochrome c release,which is the key event in the apoptotic mitochondrial signaling pathway[67].

The next possible effect of GLN supplementation on the growth of cancer cells is the modulation of IGF-I and TGF-β1concentrations in both cancer and normal cells. The proteins of insulin-like growth factor system(IGF)are known to play an important role in tumor genesis and inhibition of apoptosis.Transforming growth factor(TGF-β) is a cytokine involved in the process of cell migration,tumor vascularisation,and inhibition of cell proliferation.It is not explained if the observed effect of GLN supplementation on Bax,Bcl-2,capsace-3,IGF-I,and TGF-β1levels is the direct effect of GLN or the intermediate effect of the altered GSH metabolism.

4.Glutamine and ATP Production in Cancer Alterations in the cancer cell metabolism consist as a rule in the increased glycolysis[22],decreased TCA cycle activity [40,41],and decreased oxidative phosphorylation(OXPH) [25]in mitochondria.The TCA produces NADH2/FADH2 and OXPH uses them for ATP production.Thus,ATP production in mitochondria is often deteriorated in cancer cells and the main source of ATP remains glycolysis[22].The common feature in cancer is the overproduction of reactive oxygen species(ROS)in the OXPH chain that leads to the downregulation of the ATP production in mitochondria and to the oxidative stress[25,29–33].

The analysis of GLN metabolism presented below takes into account its lack of ability to produce ATP,especially in the context of the deteriorated ATP production via TCA cycle and OXPH chain[68].

4.1.Relation between Glutamine and Krebs Cycle.In order to investigate the effects of GLN supplementation,let us analyze the decay paths for GLN.In Figure1,let us analyze two reactions that metabolize reciprocal reactions between GLN and GLU.As discussed earlier,in the case of low ATP level in the cell,which takes place in the majority of cancers,one can expect that the equilibrium between these both reactions is strongly shifted to the left(GLU creation).

The activity of GA22in the individual types of cancers is one of important features of cancer metabolism concerning GLN[69].This enzyme is encoded by GLS and GLS2gene [70].Expression of GLS2is necessary for cells to maintain GSH levels and silencing GLS2increased ROS and oxidative damage of DNA.It is reported that gene GLS2encoding “liver-type”isozyme of GA22is highly expressed in normal adult liver but silenced in hepatocellular carcinomas[71,72].

Thus,in the case of cancers having reduced activity of GA22,

the increased oxidative stress in the cancer cells is expected.

GLN supplementation is,however,not expected to reduce

this oxidative stress and support the growth of the cancer

cells.The benefits of GLN supplementation may potentially

surpass disadvantages in this case.

Now,let us analyze relations betweenαKT and GLU.The crucial enzyme here is glutamate dehydrogenase(GDH21).As

mentioned,this reaction relies on the metabolic state of the

organism and the direction GLU→αKT dominates,due to the allosteric regulation.The activity of GDH21is inhibited

by ATP and NADH2and activated by ADP.It means that this

enzyme is active in the state of energy shortage in the cell

opening the quick energy pathway from GLN.It results in

the GLN decay and decrease in GLN levels in the blood.The

low GLN level activates the muscle proteins decay in order

to maintain the GLN levels in the blood.In the case of high

GLN utilization by the advanced cancer,this process causes

the cancer cachexia.The decay of GLN takes place,however,

also in healthy cells.In particular,the intestine epithelium

and immune system need GLN as a primary fuel and both

these systems are crucial against tumor and repair damages

after chemo-and radiotherapy[10,12,14,15,73–75].

The reactions catalyzed by AAO20,GDH21,and GA22

produce ammonia.Ammonia produced in these reactions

inside the tumor plays a crucial role in autophagy regulation

[42,56].GLN supplementation is expected to increase the

ammonia production,especially in the case of cancers with normal activity of GA22.Stimulation of autophagy denotes

potentially slower growth of the tumor and,at the same

time,possibly higher resistance to chemo-/radiotherapy,

starvation,and/or oxidative stress.

It is reported that in the case of glucose(GLC)depri-

vation,oxidation of GLN supports cell viability rather than

its growth[76].Thus,supplementation of GLN,especially

accompanied by GLC withdrawal or glycolysis-inhibition

therapy,may be treated as a potentially beneficial approach.

An inverse process(αKT→GLU→GLN)occurs especially

in muscles after the protein consumption.The reaction αKT→GLU is catalyzed by the transaminase(TA19)and it is connected with the shift of–NH2pool from other amino

acids into GLN.Thus,this pathway can take place especially

in the abundance of other amino acids that can shift its–NH2

group toαKT,as it is carried out in muscles.

4.2.Glutamine versus Pyruvate Dehydrogenase Complex (PDHC6).TCA cycle and oxidative phosphorylation (OXPH)are two factors that are essential for the effective aerobic ATP production.Figure2shows the TCA cycle reactions,the final part of the glycolysis pathway,and additional reactions in the cytoplasm that are involved in the glucose and amino acids metabolism.One of the main changes in cancer metabolism observed in majority of cancers is the deterioration of the activity of pyruvate dehydrogenase complex(PDHC6)that catalyses the reaction: pyruvate→Acetyl-CoA.This inhibition causes the reduced ATP creation[22,34,37–40].Cancers with reduced PDHC6

activity produce ATP mainly using glycolysis that is a significantly worse source of ATP than TCA+OXPH pathway.But also metabolic blocks of other TCA cycle enzymes or OXPH chain can lead to the deteriorated ATP production.Thus,the ATP level can be treated in many cases as an essential factor determining the velocity of tumor growth.Due to this observation,metabolism of the given nutrient should focus especially on its ability to support or inhibit ATP production.

In the analysis of cancer metabolism,it must be observed that one of the crucial points to be analyzed is the total number of TCA molecules in the cell(N TCA),counted both in mitochondria and in cytoplasm.N TCA is supplied by the stream of GLC and amino acids,but only one reaction is able to reduce N TCA,namely,reaction catalyzed by PDHC6. Every other reaction presented in Figure2converts only the molecules of TCA without changing its total quantity in the cell.Thus,every considered pathway of GLN degradation must go to the pyruvate.

The alternative ways of pyruvate degradation are the reactions pyruvate→lactate and pyruvate→alanine and its successive excreting outside the cell.This process takes place in many cancer types and particularly in those which are characterized by the decreased PDHC6activity.Thus,the remaining PDHC6activity,LDH4activity,and the ability to remove lactate outside the cell are crucial points determining the velocity of all metabolic paths lying before these reactions.

GLN is a molecule that must be metabolized to pyruvate. It means that,in the case of cancers with the deteriorated PDHC6,its degradation will not,in theory,occur quicker after supplementation,because GLN will“wait in line”to be metabolized by PDHC6or LDH4.The GLN-“waiting in line”is,however,not expected in healthy cells which are characterized by normal PDHC6activity.

In theory,another possibility for N TCA reduction is entering the pentose phosphate pathway by conversion to glucose-6P.This process is,however,inhibited by high level of AMP being connected with low level of ATP.Those cancers with low PDHC6activity are characterized by low ATP level since the effective energy production in mitochondria is not efficiently supplied with Acetyl-CoA.Thus,one can expect that the reduction of N TCA by pentose phosphate pathway is nondominating.

The other possibility for N TCA reduction is the conversion to some nonessential amino acid and its use in the protein synthesis.One should,however,remember that the limitation for the protein synthesis in the cancer cells depends mainly on the essential amino acids pool.The standard diet supplies both the essential and nonessential ones.GLN may support only the pool of nonessential ones.The conclusion may be drawn that this way is also not a dominating one.The possible negative influence of GLN supplementation may be expected and,however,is the case of simultaneous supplementation of GLN and essential amino acids only.Taking the essential amino acid pool into account,one can assume that the influence of GLN supplementation should be more effective if the essential amino acid pool would be simultaneously reduced in the diet.5.Utilization of GLN

The analysis of GLN utilization in cancer cells must be considered separately for the cancers possessing the reduced and normal activity of PDHC6and it must be performed especially with respect to ATP and reduced hydrogen pro-duction.Two pathways of conversion GLN→pyruvate can be considered depending on the activity of different enzymes and physiological state of the cell.They are presented in Figure3where they are called glutaminolysis(GLL)and reverse TCA(R-TCA).The basal way is GLL.Malate is the molecule that leaves mitochondria and it is next converted to pyruvate.This pathway is,however,reduced in the case of decreased activity of TCA enzymes catalyzing the reactions betweenαKT and malate that are described in some types of cancer[48,77,78].

The alternative pathway is R-TCA that consists in the conversion ofαKT to citrate.Citrate leaves mitochondria and it is converted in the cytoplasm through oxaloacetate to pyru-vate.This pathway is combined with one cycle of Acetyl-CoA pump that transports Acetyl-CoA from the mitochondria to the cytoplasm.Acetyl-CoA can be defined as the third most important(together with ATP and NADPH2)crucial nutrient for the tumor growth.This pathway is,however,from N TCA point of view,independent of Acetyl-CoA pump cycle which transports Acetyl-CoA to the cytoplasm and NADH2from cytoplasm to mitochondria.

The source for Acetyl-CoA can be the pyruvate,dietary fatty acids,and amino acids.Pyruvate can be effectively converted to Acetyl-CoA only if the activity of PDHC6is maintained.The process of Acetyl-CoA transport to the cytoplasm for the purpose of fatty and nucleic acid syn-thesis was analyzed in the subject literature[44,45].The current paper focuses rather on the importance of proportion ATP/NADPH2in cancer cell because GLN supplementation seems not to influence the Acetyl-CoA pump.The amount of Acetyl-CoA appears important only in the case of cancers having deteriorated both PDHC6activity and fatty/amino acid transport and/or their metabolism to Acetyl-CoA in mitochondria.On the other hand,it is possible that the Acetyl-CoA pump can be also deteriorated in some cases, making Acetyl-CoA the vital nutrient for the cancer growth. The crucial enzyme of Acetyl-CoA pump is cytoplasmatic ATP citrate lyase(ACL18).Its activity is often increased in cancer cells and its inhibition is proposed to be the target for cancer treatment[44,45].GLN can be the source of Acetyl-CoA only in the case of normal or close to normal activity of PDHC6[43].

5.1.Glutaminolysis.Now,let us analyze in details,two GLN→pyruvate pathways.GLN enters TCA cycle before theα-ketoglutarate dehydrogenase complex(KGDHC10).It means that the proper activity of both KGDHC10,succinyl-CoA synthetase(SCS11),succinate dehydrogenase(SDH13), and fumarate hydratase(FH14)is necessary for GLN uti-lization inside the TCA cycle.This process(glutaminolysis, GLL;see Figures2and3)is the basal pathway of GLN metabolism in the normal cells with the only exception that

the pyruvate is metabolized to Acetyl-CoA and next enters the TCA cycle.Enzymes KGDHC10and SDH13are present only in mitochondria.Thus,the proper metabolism of GLN in the GLL pathway requires proper functioning of this part of TCA cycle in mitochondria.

GLN is able to provide directly in the GLL pathway only1 molecule of ATP in the reaction of succinyl-CoA to succinate (SDH11).It is twice less than the amount of ATP produced from glucose(GLC)in the anaerobic glycolysis(2ATP/1 GLC).The remaining reactions of GLN degradation produce energy through NADH2(GDH19,KGDHC10,and MDH15ab), FADH2(SDH13),or NADPH2(GDH19,ME16).Moreover,the only molecule of ATP may not be created in the existence of the oxidative stress in cancer cell.Fedotcheva et al.[79]show that the nonenzymatic decarboxylation ofαKT(reaction12), pyruvate,and oxaloacetate induced by H2O2results in the formation of succinate,acetate,and malonate,respectively. The only molecule of ATP may not be created in this situation. It is proved that many cancer cells(lung,breast,kidney, prostate,colon,liver,skin,thyroid,and bladder)present the existence of oxidative stress according to mtDNA mutations followed by impaired TCA cycles enzyme synthesis and/or OXPH chain[29–31,33].Thus,one can assume that the nonenzymatic decarboxylation without synthesis of the only ATP molecule may occur common in cancers.

Many experiments point to the decreased activity of both different TCA enzymes and OXPH in cancer cells[29–31,33]. Blocking or reducing some of these enzymes makes it difficult to metabolize GLN in the GLL pathway.

The decreased activity of KGDHC10catalyzing the reac-tionαKT→succinyl-CoA can be expected in tumors having deteriorated activity of PDHC6since PDHC6and KGDHC10 are twin enzymatic complexes that can undergo similar regulatory processes[80].

The additional effect can be connected with the nonen-zymatic conversion of oxaloacetate to malonate.Malonate is known to be mitochondrial toxin[81]considered as the com-petitive inhibitor of SDH13and trigger of superoxide radicals [82].The malonate degradation in the cell depends on the concentration of ATP and Mg2+[83].The concentration of these two compounds is decreased in the majority of cancers causing possible further decrease in activity of this part of TCA,due to possible increased malonate concentration in the cancer cell.

It can be observed that the presented pathway of GLN decay does not produce significant ATP amounts in the cancer cells.NADH2and FADH2can be the source of ATP only after they enter the OXPH chain.The decrease in OXPH protein contents,respiratory chain activities,and mitochondrial DNA amounts in cancer are well evidenced, particularly in CCRCs[78,84–86]but also in other types of cancers[87–90].Thus,the ability of GLN to support ATP production is expected to be strongly reduced in the cancers having lowered OXPH activity.

However,in the case of cancers with OXPH chain and GLL pathway working properly,GLN supplementation may support ATP production which is generated from NADH2and FADH2in the OXPH chain.It can take place,however,

if the twin enzyme complexes(PDHC6and KGDHC10) have different activities:PDHC6is inactive and KGDHC10—

active.

Concluding,mainly cancers having deteriorated OXPH

activity orαKT→malate part of TCA cycle can be treated

as potentially surpassing the benefits over the disadvantages

while supplementing GLN.

5.2.Reverse TCA.The GLN metabolism can take place in

the case of decreased activity of KGDHC10,SCS-A11,SDH13, or FH14.GLN can be metabolized to citrate in the R-TCA

reactions(IDH9and Aconitase8).Next,citrate is transported

to the cytoplasm(see Figures2and3).Here,citrate inhibits two enzymes:phosphofructokinase(PFK1)and pyruvate dehydrogenase complex(PDHC6)—the key enzymes of gly-

colysis.Thus,in the case of cancers possessing deteriorated

GLL pathway,GLN supplementation can slow down the

glycolysis(the main source of ATP)in some types of tumor

by the agency of citrate[91].This phenomenon is mainly

expected in the case of tumors possessing deteriorated GLL

pathway which forces conversion of GLN to citrate.Next,

the citrate is metabolized in the cytoplasm by ATP citrate-lyase(ACL18)to Acetyl-CoA and oxaloacetate.This reaction

uses1molecule of ATP.Oxaloacetate can be metabolized to

malate(MDH15b)and next by using the NADP-malic enzyme (ME16)to pyruvate.Both products of ME16(NADPH2and

Acetyl-CoA)can be the substrates to fatty and nucleic acid

synthesis.The use of Acetyl-CoA for this process utilizes one

additional molecule of ATP for its activation to malonyl-CoA

[92].There is,however,also a disadvantage of this pathway. The reactions catalyzed by cytoplasmatic MDH15b and ME16

convert cytoplasmatic NADH2to NADPH2,which leads to

the additional glycolysis reaction producing NADH2without

accompanying lactate creation that produces extra2ATP

molecules.Summing up,the ATP production balance of R-

TCA pathway is close to zero.The described process occurs particularly in the case of deteriorated activity of KGDHC10, SCS-A11,SDH13,or FH14.The normal pathway of TCA cycle

cannot follow in that case.

GLL and R-TCA pathways are the only two fundamental

pathways for GLN conversion to pyruvate.The other

pathways could be considered as the connections of

one of the above defined pathways and of another

cycle of https://www.wendangku.net/doc/469836091.html,ly,if GLL pathway was joined

with Acetyl-CoA pump cycle(see Figure3),then

the following pathway could be obtained:GLN→αKT→SC→malate→oxaloac→citrate→(transport to cy-toplasm)→oxaloac→malate→pyruvate.

The analysis of GLN metabolism presented above leads

to the main conclusion that GLN cannot be,in practice,the

significant source of ATP.In the case of lowered ATP level

in the cancer cell,the supplementation of GLN does not

supply the cell significantly in ATP and the supplementation

of GLN may be beneficial rather than disadvantageous.It

should be,however,stressed that the beneficial effect is

expected particularly if the ATP reduction is supported

by accompanying glucose withdrawal in the diet and/or

glycolysis inhibition therapy that decreases ATP being a crucial metabolic substance for tumor growth.

6.Glioblastoma

All considerations presented in the former section concerned mainly cancers with the deteriorated PDHC6.Some cancers possess,however,the normal or close to normal activities of PDHC6and OXPH chain.The example is the glioblastoma multiforme(GM)[32,43]that shows normal activity of the TCA cycle and only partially deteriorated ability to produce the ATP from fatty acids in the OXPH chain[2].The activity of pyruvate kinase PC5in place of PDHC6is deteriorated in these cells.The escape of citric acid from mitochondria for fatty acid synthesis(Acetyl-CoA pump)is also observed.The oxaloacetate used for citric acid synthesis in the mitochondria is derived to a greater extent from the GLN and the Acetyl-CoA from glucose in this case.It must be observed that,due to proper activity of PDHC6,pyruvate is able to enter TCA cycle and to be the source for ATP production[93].Thus,it can be concluded that the supplementation of GLN is rather not recommended in this case because GLN can be the source of both ATP,NADPH2,and Acetyl-CoA.DeBerardinis et al.[43]showed GM by using the13C NMR spectroscopy that in the case of abundance of both GLN and GLC about 1/3of Acetyl-CoA for fatty acid synthesis comes from GLN and about2/3from GLC.This is possible due to maintained PDHC6activity.

Since the number of TCA molecules in the cell is reduced by PHDC6and not increased by PC5,GM cells need to be continuously supplied with TCA molecules for maintaining the metabolic pathways.In this case,the main source is GLN as the most important amino acid in blood that can be converted to some TCA molecule.

Many cancers lower ATP production in the case of GLC withdrawal[39,94]but not the GM[32].In the case of glucose abundance,about84%of GLC is metabolized to lactic acid,9%is metabolized to alanine,and5%is metabolized in the OXPH chain[43].This proportion reflects the typical Warburg effect.In the case of GLC withdrawal,GM stops the ATP production in the glycolysis pathway but follows the OXPH chain and the total ATP level does not decrease in the cancer cell.This process is combined with high ROS production in the OXPH chain and the GM cells start to die out due to the increased oxidative stress[32].Thus,the supplementation of GLN as the source of NADH2/FADH2 for the impaired OXPH chain producing many ROS can be potentially beneficial in this case.It should be,however, accompanied with the strong carbohydrate reduction in the diet and/or with the glycolysis inhibition therapy and/or with the increase in amino-and fatty acids in the diet that supports TCA cycle and in this way the oxidative stress in the cell.The detailed“in vivo”analysis of this approach must be,however, performed to answer if the destroying effect of the oxidative stress overcomes the tumor growth stimulation.7.Glutamine and HIF-1α

It is proved that many tumors show the overexpression of hypoxia inducible factor(HIF-1α).It is reported that HIF-1αcan be activated by a number of other oncogenes even under normoxic conditions[49].Thus,the increased activity of HIF-1αis probably a feature of many tumors.

HIF-1αis degraded by one of three different HIF prolyl hydroxylases.They are members of a superfamily of iron and α-ketoglutarate-dependent dioxygenases[95,96].The over-

expression of HIF-1αoccurs due to the inhibiting activity of accumulated succinate(SC)on the HIF prolyl hydroxylases. In other words,HIF-1αactivation is stimulated by lowered αKT/SC ratio.

The overexpression of HIF-1αleads to the increased transcription of genes encoding glycolysis enzymes like aldolase2,pyruvate kinase3,and LDH-A4[46–48].The target is also the pyruvate dehydrogenase kinase that inactivates the PDHC6.The inhibition of PDHC6by HIF-1αcauses the accumulation of pyruvate and lactate.If the removal of lactate from the cancer cell is not sufficient then the other probable consequence of this inhibition is the accumulation of preceding metabolites like malate,oxaloacetate,fumarate, and alternatively the succinate.In the case of GLN deficiency (and successive deficiency ofαKT),this situation can lead to the decreased ratio ofαKT/SC and further stabilization of HIF-1α.

GLN is a quick source ofαKT in the cancer cell.The inhibiting effect ofαKT supplementation on HIF-1αactivity is described by Matsumoto et al.[97].The in vitro antipro-liferative effect ofαKT on some kinds of tumors was also presented by Brzana et al.[98].Thus,one of the possible positive effects of GLN supplementation can be making αKT/SC ratio increased in cancer cells which can reduce the overexpression of HIF-1α.

Many other beneficial effects ofαKT supplementation are described by Harrison and Pierzynowski[99].It suggests that some of the benefits of GLN supplementation are probably obtained by the agency ofαKT.The reduction of HIF-1αshould lead to the reduction of overexpressed glycolysis and to restoration to some degree of the PDHC6activity.It is recently shown that restoration of PDHC6activity through dichloroacetate in cancer cells can promote the apoptosis of cancer cells[37].On the other hand,activation of PDHC6 can support the energy production in the cancer cells and the effect of GLN could be negative if the apoptosis caused by HIF-1αinhibition did not occur.However,the detailed analysis of this problem was not found.

8.Gluconeogenesis

Main problem related to the GLN intake by cancer patients is gluconeogenesis.Gluconeogenesis is one of the reasons for quicker GLN metabolism in cancer patients,especially in severe stages of the illness.It is known that cancers use the glucose as the main source for ATP production[22].In the case of high glucose intake by the cancer,glucose is restored in the liver from amino acids and GLN is the main source

for this process.Thus,there is a potential danger of GLN supplementation to produce glucose for ATP production in the tumor.The main aim of GLN supplementation in cancer patients is to support the immunological system, intestine tract,and glutathione synthesis and inhibit the cancer cachexia.This problem can be probably solved by experiments that will monitor the concentration of GLC and GLN in the plasma in different dietary and/or therapy conditions.Most effective doses and application time of GLN should be found in order to substantially support the organism rather than gluconeogenesis and cancer.The optimal effect of GLN supplementation is expected when it is accompanied by gluconeogenesis and/or glycolysis inhibition to slow down ATP production.Supplementation of GLN seems to make sense if the concentration of both GLN and GLC in plasma is low.GLN supports,in this case,the regen-eration of the organism without significant support of tumor growth.Support for the intestine tract(e.g.,after chemo-or radiotherapy)should be made orally,but supporting the immunological system and other healthy tissues should be made rather intravenously to omit the effect of first GLN passage through the liver.The elementary doses of GLN should be probably small enough to be consumed by healthy tissue in particular rather than converted by liver to GLC. 9.Summary

Based on the analysis of GLN metabolism,it can be concluded that if the OXPH chain is deteriorated,GLN cannot be an effective source of ATP for the cancer cell regardless of the metabolic pathway.The benefits of GLN supplementation should be probably more significant if they are accompanied by significant carbohydrate restrictions in the diet and by glycolysis and/or gluconeogenesis inhibition therapy,which will reduce the ATP level in the cancer cells that have the deteriorated mitochondria,but not in the normal cells with correctly functioning mitochondria.GLC and GLN monitoring in plasma is recommended in order to find the optimal doses and intervals of GLN that minimize gluconeogenesis effects in the liver.On the other hand, the apoptosis induction effect of GLN supplementation via HIF-1αinhibition is also possible.Supporting the intestine tract,immunological system and glutathione synthesis by GLN are especially expected to be beneficial for patients undergoing chemo-and radiotherapy.The supplementation may be highly effective after the chemo-/radiotherapy to avoid any therapy resistance effects.The other problem of GLN supplementation is its inhibition of cancer cachexia. GLN can be considered to be applied as a part of palliative care.

The dose dependent effect is expected to be strongly non-linear.In the case of small doses,the positive supplementation effect seems to depend on the reduction of GLN deficiency in healthy cells and supporting the intestine epithelium and immunological system.Medium doses are expected to support cancer metabolism rather than the organism itself. Big doses may be considered to induce HIF-1αinhibition that could activate PDHC6and,next,apoptosis.αKT supplemen-tation can be considered as an alternative approach that is expected to exhibit all effects of GLN with the exceptions concerning ammonia induced autophagy and promotion of GSH synthesis[100].

The following individual cancer metabolism features are very important in analyzing the effect of GLN supplemen-tation:PHDC6activity,individual TCA enzymes activity profile,malonate concentration,functioning of OXPH chain, oxidative stress,and HIF-1αactivity.

Abbreviations

Enzymes(The Upper Indices Point to the Reaction Numbers in Figures1and2)

AAO20:l-Amino acid oxidase

ACL18:Citrate-lyase

FH14:Fumarate hydratase

GDH21:Glutamate dehydrogenase

GA22:Glutaminase

GS23:Glutamine synthase

IDH9:Isocitrate dehydrogenase

KGDHC10:Alfa-ketoglutarate dehydrogenase complex LDH4:Lactate dehydrogenase

MDH15:Malate dehydrogenase

ME16:Malic enzyme

PC5:Pyruvate carboxylase

PDHC6:Pyruvate dehydrogenase complex

PFK1:Phosphofructokinase

SCS-A11:Succinyl-CoA synthase

SDH13:Succinate dehydrogenase

TA19:Transaminase.

Other Terms

αKT:Alfa-ketoglutarate

GLC:Glucose

GLN:Glutamine

GLU:Glutamate

GM:Glioblastoma multiforme

GSH:Glutathione

HIF-1α:Hypoxia inducible factor

OXPH:Oxidative phosphorylation

SC:Succinate

TCA:Tricarboxylic acid cycle.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

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β内酰胺类抗生素β内酰胺酶抑制剂合剂临床应用专家共识

β-内酰胺类抗生素/β-内酰胺酶抑制剂合剂临床应用专家共识 一、概述 革兰阴性菌是我国细菌感染性疾病最常见的病原体。近年来，革兰阴性菌对β-内酰胺类抗生素的耐药性不断增加，最重要的耐药机制是细菌产生各种β-内酰胺酶。β-内酰胺酶抑制剂能够抑制大部分β-内酰胺酶，恢复β-内酰胺类抗生素的抗菌活性。因此，β-内酰胺类抗生素/β-内酰胺酶抑制剂合剂在临床抗感染中的地位不断提升，已成为临床治疗多种耐药细菌感染的重要选择。目前我国临床使用的β-内酰胺类抗生素/β-内酰胺酶抑制剂合剂的种类和规格繁多，临床医师对该类合剂的特点了解不够，临床不合理使用问题较突出。为规范β-内酰胺类抗生素/β-内酰胺酶抑制剂合剂的临床应用，延缓其耐药性的发生和发展，特制定本共识。 二、主要β-内酰胺酶及β-内酰胺酶抑制剂 β-内酰胺酶是由细菌产生的能水解β-内酰胺类抗生素的一大类酶。β-内酰胺酶种类繁多，有多种分类方法，最主要的分类方法有根据β-内酰胺酶的底物、生化特性及是否被酶抑制剂所抑制的功能分类法（Bush分类法），将β-内酰胺酶分为青霉素酶、广谱酶、超广谱β-内酰胺酶、头孢菌素酶和碳青霉烯酶等；根据β-内酰胺酶末端的氨基酸序列特征的分子生物学分类法（Ambler分类法），将β-内酰胺酶分为丝氨酸酶和金属酶。目前引用较多的是基于上述2种方法建立的分类方法。见表1。 表1：β-内酰胺酶的分类和3种主要酶抑制剂的作用 功能分类分 子 分 型 主要底物 可被抑制 代表性酶 克 拉 维 酸 舒 巴 坦 他 唑 巴 坦 1 C 头孢菌素类- - - AmpC,ACT-1,CMY-2,FOX-1,MIR-1 2a A 青霉素类+ + + 青霉素酶 2b A 青霉素类，窄谱头孢菌素类+ + + TEM-1，TEM-2，SHV-1 2be A 青霉素类，超广谱头孢菌素类，单环酰胺类+ + + TEM-3,SHV-2,CTX-M-15,PER-1,VER-1 2br A 青霉素类- - - TEM-30,SHV-10,TRC-1

谷氨酰胺酶

癌症细胞中谷氨酰胺的代谢及其意义 摘要：除了加强的有氧糖酵解外，显著增加的谷氨酰胺酵解现在被认为是癌症细胞代谢特征的另一个主要特点，在这篇综述中，我们将介绍谷氨酰胺在肿瘤细胞中的主要代谢途径并阐述谷氨酰胺如何通过为肿瘤细胞提供生物代谢所需的能量和生物合成所需的前体小 分子从而维持肿瘤细胞的快速生长和增殖。最后我们重点讨论肿瘤细胞中谷氨酰胺代谢和细胞信号传导通路之间的相互影响及其在肿瘤发生发展过程中的意义。 关键词：Warburg 效应谷氨酰胺谷氨酰胺酶mTORC1(mammalian target of rapamycin) 在过去的十年中，癌症细胞的代谢作为治疗干预的靶点吸引了广泛的关注。很多癌症细胞的代谢都表现出Warburg 效应，Warburg 效应是由德国的生物化学家Otto Warburg 于1924 年首次提出，Otto Warburg 发现癌症细胞即使在正常氧分压条件下，其糖酵解代谢也非常活跃并产生大量的乳酸[1]。Warburg 效应是指在肿瘤细胞中葡萄糖摄取增加，乳酸生 成增多，细胞三羧酸循环途径产生能量减少，而利用有氧糖酵解为细胞生命活动提供能量。随后科学家们对Warburg 效应进行了深入的研究，并对癌症细胞内代谢方式的改变进行了大量的报道[2]。其中很有趣的一点是，在很多情况下，癌症细胞在表现出Warburg 效应的 同时，也对谷氨酰胺有极高的依赖性，以至于我们认为癌症细胞对谷氨酰胺成瘾[3]。谷氨酰胺代谢在肿瘤细胞中的作用及其机制已经成为当前研究的一个热点[4]。 1、谷氨酰胺代谢和谷氨酰胺酶 作为血浆中含量最丰富的氨基酸，谷氨酰胺经细胞膜上的载体转运进入细胞后进行分解代谢，在谷氨酰胺酵解过程中，谷氨酰胺进入线粒体后在谷氨酰氨。在人类基因组中有两个基因可以编码谷氨酰胺酶，谷氨酰胺酶1 基因编码肾型谷氨酰胺酶，而谷氨酰胺酶2 基因编码肝型谷氨酰胺酶[5]。肝型谷氨酰胺酶主要在肝脏中表达，而肾型谷氨酰胺酶在多种器官组织中存在表达[6]。肾型谷氨酰胺酶的在各种组织中的广泛表达使得其可能和不同类型的癌症有关。事实上，在来源于胸、肺、子宫颈、脑和B 淋巴细胞等的肿瘤中，肾型谷氨酰胺酶的表达量升高，抑制谷氨酰胺酶的活性可以抑制这些癌症细胞系的增殖[7-10]。肾型谷氨酰胺酶存在着两个转录剪切突变体，这两个突变体只在其C 端区域存在着区别，其中序列较长的称为肾型谷氨酰胺酶(KGA)，而较短的形式则被称为谷氨酰胺酶C(GAC)[11]。KGA 由谷氨酰 胺1 的第1-14 和16-19 个外显子剪切而成，而谷氨酰胺酶 1 的第二个剪切突变体谷氨 酰胺酶C（GAC）只利用了第1-15 个外显子[12]。GAC 的羧基端和KGA 不一样，并 且其蛋白分子量要比KGA小。KGA 和GAC 这两个变异体都含有完整的谷氨酰胺酶结 构域。谷氨酰胺酶C可以在很多体外组织培养的癌症细胞系中被检测到[13]。谷氨酰胺酶的这些亚型表现出不同的结构、动力学特征和组织特异性分布特点[14]。谷氨酰胺酶C 在细 胞内定位于线粒体内，而KGA 则主要存在于细胞浆中[15]。在非活化的状态下，肾型谷氨 酰胺酶和谷氨酰胺酶C 主要以二聚体的形式存在。在体外的实验中，肾型谷氨酰胺酶和谷氨酰胺酶C 都可以被无机磷酸盐活化，而无机磷酸盐的主要作用被认为是促进活化状态的谷氨酰胺酶四聚体的形成[14, 16]。

r-谷氨酰转移酶

r-谷氨酰转移酶 【原理】r－谷氨酰转移酶GGT，旧称r－谷氨酰转肽酶r－GT，它是催化谷胱甘肽上的r－谷i氨酰基转移到另一个肽或另一个氨基酸上的酶。GGT生要存在干细胞膜和微粒体上，参与谷胱甘肽的代谢。肾脏、肝脏和胰腺含量丰富，但血清中GGT主要来自肝胆系统。GGT在肝脏中广泛分布于肝细胞的毛细胆管一侧和整个胆管系统，因此当肝内合成亢进或胆汁排出受阻时，血清中GGT增高[1]。 【参考值】硝基苯酚速率法（37℃）：＜50U/L。 【临床意义】 （1）胆道阻塞性疾病：原发性胆汁性肝硬化、硬化性胆管炎等所致的慢性胆汁淤积，肝癌时由于肝内阻塞，诱使肝细胞产生多量GGT同时癌细胞也合成GGT均可使GGT明显升高，可达参考值上限的10倍以上。此时GGT、ALP、5－核苷酸酶（5－NT）、亮氨酸氨基肽酶（LAP）及血清胆红素呈平行增加。 （2）急、慢性病毒性肝炎、肝硬化：急性肝炎时，GGT呈中等度升高；慢性肝炎、肝硬化的非洁动期，酶活性正常，若GGT持续升高，提示病变洁动或病情恶化。 （3）急、慢性酒精性肝炎、药物性肝炎：GGT可呈明显或中度以上升高（300～1000U/L），ALT和AST仅轻度增高，甚至正常。酗酒者当其戒酒后GGT可随之下降。 （4）其他：脂肪肝、胰腺炎、胰腺肿瘤、前列腺肿瘤等GGT亦可轻度增高。 ★★★下面是参考： 谷草转氨酶在心肌细胞中含量最高，但肝脏损害时其血清浓度也可升高，临床一般常作为心肌梗塞和心肌炎的辅助检查。谷草转氨酶的正常值为0～40μ/L，当谷丙转氨酶（ALT）明显升高，谷丙/谷草比值＞1时，就提示有肝实质的损害。 γ-谷氨(酰转肽酶（γ-GT）广泛分布于人体组织中，肾内最多，其次为胰和肝，胚胎期则以肝内最多，在肝内主要分布于肝细胞浆和肝内胆管上皮中，正常人血清中γ-GT主要来自肝脏。正常值为3～50μ/L（γ-谷氨酰对硝基本胺法）。此酶在急性肝炎、慢性活动性肝炎及肝硬变失代偿时仅轻中度升高。但当阻塞性黄疸时，此酶因排泄障碍而逆流入血，原发性肝癌时，此酶在肝内合成亢进，均可引起血中转肽酶显著升高，甚至达正常的10倍以上。酒精中毒者ν-GT亦明显升高，有助于诊断酒精性肝病。在急性肝炎时，ν-GT下降至正常较转氨酶为迟，如ν-GT持续升高，提示转为慢性肝病。慢性肝病尤其是肝硬化时，ν-GT持续低值提示预后不良

TG谷氨酰胺转胺酶性质及其在肉制品中的应用说明

谷氨酰胺转胺酶性质及其在肉制品中的应用说明 一、谷氨酰胺转胺酶（TGase ）介绍 谷氨酰胺转胺酶（Transglutaminase ，简称TG 、TGase 、mTG 等），又称转谷氨酰胺酶，是一种酰基转移酶，能够促进蛋白质分子内交联、蛋白质分子间交联以及蛋白质和氨基酸之间的交联。TGase 能够催化蛋白质中谷氨酰胺残基的γ-酰胺基和赖氨酸的ε-氨基之间进行酰胺基转移反应，形成ε-（γ-谷酰胺）-赖氨酸的异型肽键，改善蛋白质的功能性质，可以有效地提高蛋白性食品的弹性、持水能力、原料利用率、质地口感及营养价值等，现已广泛应用于肉制品、乳制品、鱼制品、面制品、豆制品等。 谷氨酰胺转胺酶广泛存在于自然界中，早期TGase 是从动物肝脏中提取，成本较高，应用受到限制。本公司采用现代生物工程发酵技术，利用微生物法发酵生产并精制提取而成，具有酶活高、催化效率高等特点。1.1TGase 的结构： 1.2TGase 的催化机理 TGase 利用肽链上的谷氨酰胺残基上的甲酰胺基为乙酰基供体，受体可以是蛋白质上的或游离氨基酸上的胺基、伯胺基、水。TGase 既可以催化蛋白分子间的交联，又可以催化分子内的交联反应。TGase 催化的主要反应 如下： 注： a 酰基转移反应 b 蛋白质Gln 残基和Lys 残基之间的交联反应 c 脱氨基化反应二、谷氨酰胺转胺酶（TGase ）酶学性质 1.最适pH 2.pH 稳定性 活性中 心

*谷氨酰胺转胺酶在pH 5-8的范围内具有很高的活性，最适pH 为6-7，在一般的食品加工过程中不会发生酶失活问题。*谷氨酰胺转胺酶在pH 值5-8的范围内具有很好的稳定性，当pH 低于5时，酶活迅速降低，当pH 高于8小于9时，酶活下降缓慢。3.最适温度 *谷氨酰胺转胺酶可在5℃-60℃的温度条件下发挥作用，最佳使用温度为50℃，在45℃-55℃范围内均具有较好的活性。 4.温度稳定性 *谷氨酰胺转胺酶在温度低于40℃时保持稳定，50℃以上酶活稍有下降，当温度高于75℃时酶失去活性。 5.反应温度和时间关系 *在温度不高于最适温度50℃情况下，反应时间随反应温度的升高而降低。（不同温度下的反应时间均在pH6.0条件下测定） 6.香肠内部温度和失活时间关系 温度（℃) 时间652h 以上7015min 之内755min 之内80 1min 之内 *对直径为3cm 的香肠，酶失活所需要的加热时间，每根香肠达到指定温度用冰水迅速冷却。由上表检测结果可见，酶失活所需要的时间取决于内部中心的温度，内部温度越高，失活也越快。 三、谷氨酰胺转胺酶（TGase ）的使用方法 掌握正确的TG 使用方法，对于TG 的作用发挥具有重要作用，根据TG 在催化蛋白质反应中的规律及TG 的酶学性质，谷氨酰胺转胺酶的使用方法主要有以下三种：1..溶液法 把1份谷氨酰胺转胺酶（TG ）放入3～3.5倍的水中溶解后，将水溶液加到肉中、充分搅拌、装模成型，经过一段时间的酶反应，使肉块粘在一起，本品一旦与水溶解后，必须在20-30分钟内与肉块搅拌并成型。2.和盐水一起加入 肉制品能快速吸收盐水，谷氨酰胺转胺酶（TG ）可先放到盐水溶液中，然后一起加入肉制品中，进行浸泡，充分混合，并在20-30分钟内成型。3.涂粉法 对于浸泡过的或已加入溶液的肉制品，谷氨酰胺转胺酶（TG ）可以直接以干粉形态加入。加入时，必须搅拌或翻转，使所有肉制品表面涂粉均匀，加入至涂粉成型必须在20-30分钟内完成。四、谷氨酰胺转胺酶（TGase ）的应用举例1.谷氨酰胺转胺酶（TG ）在肉产品中的应用 谷氨酰胺转胺酶是一种催化酰醛转移反应的酶，它能够通过形成蛋白质分子间共价键，催化蛋白质分子聚合和交联。TG 以肉制品蛋白质肽链上的谷氨酰胺残基中的甲肽氨基为供体，赖氨酸残基中的氨基为受体，催化转氨基反应，从而使蛋白质分子内或分子间发生交联。 TG 具有pH 稳定性好，热稳定性高，粘合性极强等特性，形成的共价键在非酶催化条件下（如冷冻、切片、烹饪）很难断裂，使用安全。

β-内酰胺类抗生素β内酰胺酶抑制剂复方制剂临床应用专家共识(2020年版)

β-内酰胺类抗生素β内酰胺酶抑制剂复方制剂临床应用 专家共识（2020年版） 一、概述 革兰阴性菌及少数革兰阳性菌对β-内酰胺类抗生素耐药的最重要机制是产生各种β-内酰胺酶。β-内酰胺酶抑制剂能够抑制部分β-内酰胺酶，避免β-内酰胺类抗生素被水解而失活。因此，β-内酰胺类抗生素/β-内酰胺酶抑制剂复方制剂（简称β-内酰胺酶抑制剂复方制剂）是临床治疗产β-内酰胺酶细菌感染的重要选择。我国临床使用的β-内酰胺酶抑制剂复方制剂的种类和规格繁多，临床工作者对该类制剂的特点了解参差不齐，临床不合理使用问题比较突出。 二、主要β-内酰胺酶及产酶菌流行情况 β-内酰胺酶是由细菌产生的，能水解β-内酰胺类抗生素的一大类酶。β-内酰胺酶种类繁多，有多种分类方法，最主要的分类方法有两种： 一、是根据β-内酰胺酶的底物、生化特性及是否被酶抑制剂所抑制的功能分类法（Bush分类法），其将β-内酰胺酶分为青霉素酶、广谱酶、超广谱β-内酰胺酶（ESBLs）、头孢菌素酶（AmpC酶）和碳青霉烯酶等； 二、是根据β-内酰胺酶末端的氨基酸序列特征的分子生物学分类法（Ambler分类法），将β-内酰胺酶分为丝氨酸酶（包括A类、C类酶和D 类酶）及金属酶（B类酶）。目前引用较多的是1995年Bush等基于上述二种方法建立的分类方法，2019年Bush等又将该分类表进一步完善和细化（表1）。其中临床意义最大的是下列三类β-内酰胺酶： 表1 常见β-内酰胺酶分类及特点，常见酶抑制剂抑酶活性

1、ESBLs主要属2be\2br\2ber类酶，是由质粒介导的能水解青霉素类、头孢菌素及单环酰胺类等β-内酰胺类抗生素的β-内酰胺酶，其对碳青霉烯类和头霉素类水解能力弱。ESBLs主要由肠杆菌科细菌产生，以肺炎克雷伯菌、大肠埃希菌、变形杆菌最为常见。根据编码基因的同源性，ESBLs可分为TEM型、SHV型、CTX-M 型、OXA型和其他型共5大类型。 2、AmpC酶属C类酶，通常由染色体介导，可以被β-内酰胺类抗生素诱导。部分由质粒介导，常呈持续高水平表达。其对第一、二、三代头孢菌素水解能力强，但对碳青霉烯类抗生素和第四代头孢菌素的水解能力弱。该酶主要存在于肠杆菌属、柠檬酸杆菌属、普鲁菲登菌属、黏质沙雷菌属和摩根菌属等细菌，非发酵菌中主要见于铜绿假单胞菌。质粒介导的β-内酰胺酶可分为CMY-2组、CMY-1组、MIR-1/ACT-1组、DHA-1组和ACC-1组等。 3、碳青霉烯酶是指能水解碳青霉烯类抗生素的一大类β-内酰胺酶，分别属于Ambler分子分类中的A类、B类和D类酶。A类、D类为丝氨酸酶，B类为金属酶，以锌离子为活性中心。A类碳青霉烯酶可由染色体介导，也可由质粒介导。前者包括SME、NMC和IMI酶等，后者包括KPC和GES酶等。KPC酶是近年来肠杆菌科细菌尤其是肺炎克雷伯菌对包括碳青霉烯类抗生素在内的几乎所有β-内酰胺类抗生素耐药的最主要机制，我国最常见的是KPC-2，其对头孢吡肟和头孢他啶的水解能力相对较弱。

y谷氨酰转移酶偏高是怎么一回事

y谷氨酰转移酶偏高是怎么一回事 首先大家要知道y谷氨酰转移酶，是什么意思它是反映自己肝功能的重要指标。所以大家一定要保护肝脏这些器官，如果保护不当被病毒感染时就会使y谷氨酰转移酶偏高。所以大家一定要注意平时的一些小，当一些良好的小习惯养成的时候也是对自己身体的一种保护。那么现在就让我们来了解一下y谷氨酰转移酶偏高危害吧。 γ-谷氨酰转肽酶简称γ-GT或GGT。它存在于肾、胰、肝、脾、肠、脑、肺、骨骼肌和心肌等组织中，在肝内主要存在于肝细胞浆和肝内胆管上皮中。GGT对各种肝胆疾病均有一定的临床价值。在大多数肝胆疾病中，其活力均升高，但在不同的肝胆疾病中，其升高的程度与其他血清酶活性的相对比例不尽相同。γ-谷氨酰转肽酶(GGT) 是一种方便的、高敏感的、低花费、常用的检测酶类之一。它作为肝脏功能异常和酗酒的标志已广泛的被临床接受，主要用于诊断肝胆疾病。

γ-谷氨酰转肽酶(GGT)，临床意义，肝脏疾病，酒精肝 人体各组织均含有GGT，尤以分泌或吸收能力强的富含蛋白质的组织中最多，如胆管、肾脏、胰腺、附睾等。人体各器官中GGT含量按下列顺序排列：肾、前列腺、胰、肝、盲肠和脑。而在肾脏、胰腺和肝脏中，此酶的含量之比约为100：8：4(1)。肝脏含酶丰富,酶参与肝脏复杂的代谢功能,由于肝脏病理改变,使酶含量改变,故反映在血清中酶的浓度也有变化,根据血清酶活性的增高或降低,可判断肝脏病变的性质和程度。人血清中的γ谷氨酰转肽酶主要来自肝脏，因此具有较强的特异性。在肝功能检查中，经常会出现γ谷氨酰转肽酶偏高的现象，为探讨引起γ谷氨酰转肽酶偏高的原因，和对肝功能的影响。 现在大家都知道y谷氨酰转移酶偏高的危害了，所以大家一定要注意自己的一些小习惯。不要随便的暴饮暴食，这样的习惯只会一时爽后悔终身。可见平时的生活中许多的小习惯，看着是个小问题随时都会变成一个大问题。所以大家一定要养成一些良好的习惯，这样对做任何事都有好处。相信通过大家的努力，自己身体都会健康的!

转谷氨酰胺酶

人转谷氨酰胺酶2C多肽（TGM2）酶联免疫分析试剂盒 使用说明书 本试剂盒仅供体外研究使用! 预期应用 ELISA法定量测定人血清、血浆或其它相关生物液体中TGM2含量。 实验原理 用纯化的抗体包被微孔板，制成固相载体，往包被抗TGM2抗体的微孔中依次加入标本或标准品、生物素化的抗TGM2抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的TGM2呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。 试剂盒组成及试剂配制 1.酶联板：一块（96孔） 2.标准品（冻干品）：2瓶，每瓶临用前以样品稀释液稀释至1ml，盖好后静置10分钟以上，然后反复颠倒/搓动以助溶解，其浓度为10ng/ml，做系列倍比稀释（注：不要直接在板中进行倍比稀释）后，分别稀释成10ng/ml，5ng/ml，2.5ng/ml，1.25ng/ml，0.625ng/ml，0.312 ng/ml，0.156ng/ml，样品稀释液直接作为标准浓度0ng/ml，临用前15分钟内配制。如配制5ng/ml标准品：取0.5ml（不要少于0.5ml）10ng/ml的上述标准品加入含有0.5ml样品稀释液的Eppendorf管中，混匀即可，其余浓度以此类推。 3.样品稀释液：1×20ml。 4.检测稀释液A：1×10ml。 5.检测稀释液B：1×10ml。 6.检测溶液A：1×120μl（1:100）临用前以检测稀释液A1:100稀释，稀释前根据预先计算

好的每次实验所需的总量配制（100μl/孔），实际配制时应多配制0.1-0.2ml。如10μl检测溶液A加990μl检测稀释液A的比例配制，轻轻混匀，在使用前一小时内配制。 7.检测溶液B：1×120μl/瓶（1:100）临用前以检测稀释液B1:100稀释。稀释方法同检测溶液A。 8.底物溶液：1×10ml/瓶。 9.浓洗涤液：1×30ml/瓶，使用时每瓶用蒸馏水稀释25倍。 10.终止液：1×10ml/瓶（2N H2SO4）。 11.覆膜：5张 12.使用说明书：1份 自备物品 1.酶标仪（建议参考仪器使用说明提前预热） 2.微量加液器及吸头，EP管 3.蒸馏水或去离子水，全新滤纸 标本的采集及保存 1.血清：全血标本请于室温放置2小时或4℃过夜后于1000x g离心20分钟，取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻融。 2.血浆：可用EDTA或肝素作为抗凝剂，标本采集后30分钟内于2-8°C1000x g离心15分钟，或将标本放于-20℃或-80℃保存，但应避免反复冻融。 3.其它生物标本：请1000x g离心20分钟，取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻融。 4.样本处理：血清或血浆标本推荐稀释10倍，如：稀释10倍，取100uL血清或血浆加入900uL 样品稀释液。标本使用0.1M的PBS稀释（PH=7.0-7.2）。 注：以上标本置4℃保存应小于1周，-20℃或-80℃均应密封保存，-20℃不应超过1个月，-80℃不应超过2个月；标本溶血会影响最后检测结果，因此溶血标本不宜进行此项检测。

谷氨酰胺酶(glutaminase, GLS)活性测定试剂盒使用说明

谷氨酰胺酶(glutaminase,GLS)活性测定试剂盒使用说明 产品简介： GLS存在于高等动物和某些细菌以及植物根中，催化谷氨酰胺水解成谷氨酸和氨，在氮素代谢中具有重要调控作用，尤其是调节游离氨含量和尿素代谢。GLS催化谷氨酰胺水解成L-谷氨酸和氨，利用奈氏试剂检测氨增加的速率，即可计算其酶活性。 试验中所需的仪器和试剂： 台式离心机、可见分光光度计、1mL玻璃比色皿、可调式移液枪、研钵、冰和双蒸水。 产品内容： 试剂一×1瓶，60mL，4℃保存； 试剂二×1瓶，50mL，4℃保存； 试剂三×1瓶，60mL，常温保存； 试剂四×1瓶，12mL，常温保存； 试剂五×1瓶，6mL，常温保存； 试剂六×1瓶，6mL，常温避光保存。 操作步骤： 一、粗酶液提取： 称取约0.1g组织，加入0.9ml试剂一研钵中研磨均匀，于48000rpm℃离心10min，取上清，作为待测液（粗酶液）。

二、测定步骤： 1、空白管： （1）蒸馏水0.05mL，加试剂一0.2mL，加试剂二0.8mL，混匀后37℃水浴1h； （2）加入试剂三1.05mL，混匀后8000rpm离心10min；取上清液1.0mL到新离心管中，加入试剂四0.23mL，混匀； （3）取0.8mL到新离心管中，依次加入试剂五0.1mL和试剂六0.1mL，混匀后等待10min，即可用于调零。 2、样品管：仅需要把蒸馏水0.05mL换成粗酶液0.05mL即可，其余同空白管。 3、实际测定只要做一个空白管，用于调零，于420nm处比色，记录吸光值，记为A。 GLS活性计算： 1、酶活性单位定义：37℃下每mg蛋白质每小时催化谷氨酰胺生成1μmol氨。 2、计算公式： GAA（U/mg prot）＝363.1×(A－0.1301)÷蛋白质浓度（mg/mL）。

谷氨酰转移酶测定

谷氨酰转移酶测定 1 检验目的 指导本室工作人员规范操作本检测项目，确保检测结果的准确。 2 实验原理 L-γ-谷氨酰-3羧基-4硝基苯胺 + 甘氨酰甘氨酸GGT L-γ-谷氨酰甘氨酰甘氨酸 + 5-氨基-2-硝基苯甲酸盐 在上述反应中， 5氨基2硝基苯甲酸盐的生成速率与样本中γ谷氨酰基转移酶的活力成正比，通过在405 nm处监测吸光度的上升速率，即可测得样本中γ谷氨酰基转移酶的活性。 3 标本： 3.1 病人准备：无特殊。 3.2 类型：血清或EDTA血浆。 3.3 标本存放：室温可保存8小时；2～8℃可保存3天；-20℃保存至少可稳定1周。

3.4 标本运输：常温条件下保存运输。 3.5 标本拒收标准：细菌污染的标本。 4 实验材料 4.1 试剂：上海复星长征医学科学有限公司GGT试剂盒（沪食药监械（准）字2014第2400166号 YZB/沪 1546-40-2014）4.1.1 试剂组成 试剂1（R1）：Tris缓冲 液100mmol/ L 甘氨酰甘氨酸 125mmol/L 试剂2（R2）：Tris缓冲 液100mmol/ L L-γ-谷氨酰 -3羧基-4硝基 苯胺 14.5mmol/L 4.1.2 试剂准备：试剂为即用式。 4.1.3 试剂稳定性与贮存:2～8℃避光、密封的储存条件下，试剂（盒）自生产之日起有效期为12个月。 4.1.4 变质指示：当试剂有看得见的微生物生长，有浊度，或者未开盖的液体有沉淀时，表明试剂已变质，不能继续使用。

4.1.5 注意事项：试剂中含叠氮钠为防腐剂。不可入口！避免接触皮肤及粘膜。应采取必要的预防措施使用试剂。 4.2 校准品：使用上海复星长征医学科学有限公司提供的GGT校准品对自动分析仪进行校准。 4.3 质控品：使用正常值、病理值复合控制品。 5 仪器 AU2700生化分析仪，罗氏P800生化分析仪, 西门子ADVIA-2400生化分析仪，东芝TBA-120生化分析仪 6 操作步骤 6.1 样品的准备：将标好号的样品离心后放到仪器规定的位置。 6.2 试剂的检测：仪器开机后，检查各种试剂的位置，体积等确认无误后方可进行测定。 6.3 项目基本参数：参见生化检验AU2700生化分析仪，罗氏P800生化分析仪, 西门子ADVIA-2400生化分析仪，东芝TBA-120生化分析仪项目测定参数。

转谷氨酰胺酶在肉制品中的应用研究

转谷氨酰胺酶在肉制品中的应用研究 转谷氨酰胺酶学名谷氨酰胺转胺酶，是一种非常优良的蛋白质改良剂，能够催化蛋白质分子内、分子间连接交联，蛋白质和氨基酸之间的连接以蛋白质分子内谷氨酰胺基的水解，同时改善蛋白质的功能和性质。我国肉类总产量位居全球第一，但肉制品量只占总量的6%，从发达国家的肉制品量占据肉类总产量的40%～50%来看，说明我国在肉类加工领域肉还有很大潜力，转谷氨酰胺酶作为一种优良的肉制品加工改良剂，在肉制品方面有巨大的应用潜力。 谷氨酰胺转 转谷氨酰胺酶广泛分布于自然界的生物中，转谷氨酰胺酶在豚鼠肝脏中被卡拉克人等首次发现，经过不断的研究发现微生物、植物和其他动物中也存在转谷氨酰胺酶。 动物来源哺乳动物几乎所有的组织和器官中都存在转谷氨酰胺酶。20世纪60年代到90年代，从豚鼠肝脏中提取转谷氨酰胺酶，由于来源较少，并且纯化工艺复杂，这两方面导致价格昂贵。 植物来源1987年，艾斯卡森等人发现转谷氨酰胺酶存在于豌豆中。研究者已经发现在马铃薯、菊芋、玉米等多种植物中也有转谷氨酰胺酶的存在。有研究者对大豆中提取的转谷氨酰胺酶进行深入研究，但是发现分离和纯化工艺非常复杂，酶得率也相对较低。所以迄今为止，植物来源的转谷氨酰胺酶还没有用于商业化生产。 微生物来源1989年，安腾等人首次从茂原链轮丝细菌中首次分离并且纯化得到了转谷氨酰胺酶。随后，研究人员在其他微生物中发现了转谷氨酰胺酶存在，并进行大量的微生物发酵试验、酶纯化分离试验等，取得了很大的进步。与动物来源转谷氨酰胺酶相比，微生物来源转谷氨酰胺酶属于胞外酶，它能够在培养基中直接分泌，分离和纯化相对比较容易，发酵所用的原料价格较低、生产周期短，最有前途用于大规模的工业化生产。 转谷氨酰胺酶的理化性质 不同来源的转谷氨酰胺酶其理化性质差异动物肝脏的转谷氨酰胺酶的分子量大约在70～90kDa之间，需要通过钙的离子激活来实现其功能性，酶的活性中心为半胱胺酸铵残基位点。动物来源转谷氨酰胺酶中，豚鼠肝脏的转谷氨酰胺酶研究最为深入，研究发现酶分子量大约为90KDa，使酶反应需要钙离子去激活，底物特异性较强，同时该酶含有多个半胱氨酸残基导致其热稳定性较差，50℃保持10min其酶活性仅为残留的40%。 与动物的来源相比，微生物来源的转谷氨酰胺酶具有更加优良的特性：一是具有较低的分子量，分子量在23000～45000，多数为40000左右，高度的交联催化度；二是具有更强的耐热性，来源于S．mobaraensis的转谷氨酰胺酶在40℃条件下，即使保持10min，酶仍然具有相同的活性，即使在50℃的条件下，保持10min该酶仍具有74%的活性；三是耐酸耐碱性强；四是是否存在钙离子对酶的活性影响不大，这一特性非常重要，因为绝大多数的蛋白质容易与钙离子发生化学反应，导致蛋白质沉淀；五是转谷氨酰胺酶耐高温高压，周围

21、谷氨酰氨基转移酶标准操作规程(GGT)

前言 为使临床检验操作规范化，指导检验人员严格按规程进行正确的常规操作，保证检验质量，特制定本操作规程。本规程的编写遵循了ISO 15189《医学实验室——关于质量和能力的特殊要求》及WS/T 227-2002《临床检验操作规程编写要求》的有关规定并结合产品实际情况制订，作为本产品的标准操作程序。 本规程从2007年5月2日起实施，每2年复审1次。 本规程由浙江伊利康生物技术有限公司编制。 本规程起草单位：伊利康生物技术有限公司技术部。 本规程主要起草人：蒙凯、蔡其浩。 本规程首次起草。

目录 1 检验申请 (3) 2 标本采集与处理 (3) 3 试剂及成份 (4) 4方法原理 (4) 5 仪器 (4) 6 校准液及校准模式 (4) 7质控品与室内质控规则 (4) 8标本检测步骤 (5) 9 结果计算 (5) 10 操作性能 (5) 11试剂使用的注意事项 (5) 12参考范围及医学决定水平 (5) 13检验结果的报告及范围 (5) 14临床意义 (6) 15结果审核分析以及相关项目的联系 (6) 16威胁生命的“紧急值”及报告规定. (6) 17有关引用程序与文件 (6) 18参考文献 附录A XXX型全自动生化分析仪参数

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详解β-内酰胺类抗生素和β-内酰胺酶抑制剂

详解β-内酰胺类抗生素和β-内酰胺酶抑制剂 详解β-内酰胺类抗生素和β-内酰胺酶抑制剂 一、概述 革兰阴性菌是我国细菌感染性疾病最常见的病原体。近年来，革兰阴性菌对β-内酰胺类抗生素的耐药性不断增加，最重要的耐药机制是细菌产生各种β-内酰胺酶。β-内酰胺酶抑制剂能够抑制大部分β-内酰胺酶，恢复β-内酰胺类抗生素的抗菌活性。因此，β-内酰胺类抗生素/β-内酰胺酶抑制剂合剂在临床抗感染中的地位不断提升，已成为临床治疗多种耐药细菌感染的重要选择。目前我国临床使用的β-内酰胺类抗生素/β-内酰胺酶抑制剂合剂的种类和规格繁多，临床医师对该类合剂的特点了解不够，临床不合理使用问题较突出。为规范β-内酰胺类抗生素/β-内酰胺酶抑制剂合剂的临床应用，延缓其耐药性的发生和发展，特制定本共识。 二、主要β-内酰胺酶及β-内酰胺酶抑制剂 β-内酰胺酶是由细菌产生的能水解β-内酰胺类抗生素的一大类酶。β-内酰胺酶种类繁多，有多种分类方法，最主要的分类方法有根据β-内酰胺酶的底物、生化特性及是否被酶抑制剂所抑制的功能分类法（Bush分类法），将β-内酰胺酶分为青霉素酶、广谱酶、超广谱β-内酰胺酶、头孢菌素酶和碳青霉烯酶等；根据β-内酰胺酶末端的氨基酸序列特征的分子生物学分类法（Ambler分类法），将β-内酰胺酶分为丝氨酸酶和金属酶。目前引用较多的是基于上述2种

方法建立的分类方法。 超广谱β-内酰胺酶（ESBLs）是由质粒介导的能水解青霉素类、头孢菌素及单环酰胺类等β-内酰胺类抗生素的β-内酰胺酶，其对碳青霉烯类和头霉素类水解能力弱。这类酶可被β-内酰胺酶抑制剂如克拉维酸、舒巴坦及他唑巴坦等抑制。ESBLs主要由肠杆菌科细菌产生，以肺炎克雷伯菌、大肠埃希菌、变形杆菌最为常见。到目前为止，全世界共发现了200余种ESBLs。根据编码基因的同源性，ESBLs可分为TEM型、SHV型、CTX-M型、OXA型和其他型共5大类型。 头孢菌素酶（AmpC酶）通常是由染色体介导，对第一、二、三代头孢菌素水解能力强，但其对碳青酶烯类抗生素和第四代头孢菌素的水解能力弱，克拉维酸钾不能抑制其活性，他唑巴坦和舒巴坦有部分抑酶作用，氯唑西林抑制头孢菌素酶作用强。该酶主要存在于肠杆菌属、柠檬酸杆菌属、普鲁菲登菌属、粘质沙雷菌属和摩根菌属等细菌。染色体介导的头孢菌素酶可以被β-内酰胺类抗生素诱导和选择。近年来，质粒介导的头孢菌素酶陆续被报道，主要出现于肺炎克雷伯菌、大肠埃希菌及沙门菌属细菌中，常呈持续高水平表达，可通过质粒广泛传播。根据其与染色体介导的头孢菌素酶的同源性，可分为CMY-2组、CMY-1组、MIR-1/ACT-1组、DHA-1组和ACC-1组等。 碳青霉烯酶是指能水解碳青霉烯类抗生素的一大类β-内酰胺酶，分别属于Ambler分子分类中的A类、B类和D类酶。A类、D类为丝氨酸酶，B类为金属酶，以锌离子为活性中心。 A类碳青霉烯酶可以由染色体介导，也可由质粒介导，前者包括

r谷氨酰基转移酶偏高是由什么引起的

如对您有帮助，可购买打赏，谢谢 r谷氨酰基转移酶偏高是由什么引起的 导语：大家都知道，酶的作用之一就是催化，r谷氨酰基转移酶与肝胆有着非常密切的关系，主要来衡量肝胆的情况。r谷氨酰基转移酶偏高，最有可能的原 大家都知道，酶的作用之一就是催化，r谷氨酰基转移酶与肝胆有着非常密切的关系，主要来衡量肝胆的情况。r谷氨酰基转移酶偏高，最有可能的原因是你胆管受阻或是患了慢性肝炎。要想快速使这种酶降低，饮食上要忌烟酒，不要熬夜加班。酶的本质是蛋白质，因此不要过多摄入高蛋白的食物。 r谷氨酰转移酶偏高的原因是什么？哪些原因会引起谷氨酰基转移酶偏高呢？常见的病理性因素：谷氨酰转移酶偏高的原因一：原发性肝硬化、肝癌时由于肝内阻塞，肝脏损伤，从而导致谷氨酰转移酶升高，肝癌时期谷氨酰转移酶升高达到高峰。谷氨酰转移酶偏高的原因二：急、慢性病毒性肝炎、肝硬化时，谷氨酰转移酶都会升高。如果慢性肝炎、肝硬化患者属于非活动期，若谷氨酰转移酶升高，则表明病情恶化。 非病理性因素：谷氨酰转移酶偏高的原因一：对于长期饮酒，造成脂肪肝，肝脏受损，肝功能下降，从而导致谷氨酰转移酶会有明显的升高现象。谷氨酰转移酶偏高的原因二：由于饮食不当形成的脂肪肝、胰腺炎、胰腺肿瘤等也同样会引起谷氨酰转移酶升高。谷氨酰转移酶偏高的原因三：20～30岁超负荷的男性谷氨酰氨基转移酶升高约58%。50～60岁男、女性谷氨酰氨基转移均约升高30%。慢性酒精中毒者谷氨酰氨基转移酶约升高20%～400%。90kg～40kg的女性谷氨酰氨基转移酶可升高42%。4～10岁的儿童谷氨酰氨基转移酶约降低10%。 谷氨酰转移酶偏高的原因四：对于女性安眠药可导致谷氨酰氨基转 生活常识分享

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表1常见B-内酰胺酶分类及特点，常见酶抑制剂抑酶活性

1、E SBLs主要属2be\2br\2ber 类酶，是由质粒介导的能水解青霉素类、头抱 菌素及单环酰胺类等B -内酰胺类抗生素的B -内酰胺酶，其对碳青霉烯类和头霉素类水解能力弱。ESBLs主要由肠杆菌科细菌产生，以肺炎克雷伯菌、大肠埃希菌、变形杆菌最为常见。根据编码基因的同源性，ESBLs可分为TEM型、SHV型、 CTX-M型、OXA型和其他型共5大类型。 2、A mpC酶属C类酶，通常由染色体介导，可以被B -内酰胺类抗生素诱导。部分由质粒介导，常呈持续高水平表达。其对第一、二、三代头抱菌素水解能力强，但对碳青霉烯类抗生素和第四代头抱菌素的水解能力弱。该酶主要存在于肠杆菌 属、柠檬酸杆菌属、普鲁菲登菌属、黏质沙雷菌属和摩根菌属等细菌，非发酵菌

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谷氨酰胺转氨酶在烘培食品加工中的作用 导读：烘焙食品由于其营养、美味、方便、实惠而深受人们的喜爱。但是由于我国面包专用粉的质量稳定性差以及面包制作过程中机械搅拌力的破坏，导致面筋的筋力不足，从而影响面包品质。使用面包改良剂是提高面包品质的一个重要手段。酶制剂作为天然来源的面包改良剂，越来越受到青睐。 什么是谷氨酰胺转胺酶 谷氨酰胺转胺酶(简称TGase)是一种催化酰基转移反应的转移酶，它能够促使蛋白质分子内交联、分子间交联以及蛋白质和氨基酸之间交联。可以在很大程度上改善蛋白质的功能性质。 谷氨酰胺转胺酶作用 1添加TGase后，小麦粉的吸水率略有提高。这是由于TGase具有很高的亲水性，使得面团的吸水率有所增加。面团的形成时间和稳定时间有所提高。稳定时间越长，韧性越好，面筋的强度越大，面团的加工性质越好。 2添加TGase后，小麦粉的弱化度显著减小。弱化度表明面团的耐破坏程度，也就是对机械搅拌的承受能力，弱化度越大，表明该小麦粉的面筋越弱，面团越容易流变，成品不易成型，且易塌陷。弱化度减小，面筋网络结构和耐机械搅拌能力得到增强，小麦粉的粉质特性得到改善。 3添加TGase后，使得蛋白质分子间和分子内的交联作用得到加强，从而增强了面筋的网络结构和面团的稳定性。同时面包的体积和比容均有所增大。 4添加TGase后，面包的持水性得到提高。水分的保持有效抑制了淀粉的老化，面包的硬度有所减小，面包的弹性明显增大。贮藏过程中老化焓值减小，有效抑制了面包的老化，延长了面包的货架期。 小提示 食品酶制剂以其高效、安全等优点广泛应用于面包生产中。小麦粉中加入适量的谷氨酰胺转氨酶，可改善面团的粉质特性、拉伸特性和流变学特性，增大面包的体积和比容，提高了面包的持水性，改善面包质构，抑制了淀粉的老化，有效延长了面包的货架期。

谷氨酰胺转氨酶

谷氨酰胺转氨酶 谷氨酰胺转氨酶 英文名称：Glutamine transaminase 别名：转谷氨酰胺酶 产品说明：谷氨酰胺转胺酶(Microbialtransglutaminase ) 是一种催化蛋白质间（或内）酰基转移反应,从而导致蛋白质(或多肽) 之间发生共价交联的酶 谷氨酰胺转氨酶又称转谷氨酰胺酶（TG酶）是由331个氨基组成的分子量约38000的具有活性中心的单体蛋白质，其可催化蛋白质多肽发生分子内和分子间发生共价交联，从而改善蛋白质的结构和功能，对蛋白质的性质如：发泡性，乳化性，乳化稳定性，热稳定性、保水性和凝胶能力等效果显著，进而改善食品的风味、口感、质地和外观等。传统肉类加工工艺通常加入大量的盐和磷酸，以提高其持水力、连贯性和质地。近期，少盐少磷酸的食物被广泛推广，但其质地和物理性质都不尽如人意。TG酶可以替代部分通常肉制品加工中添加的品质改良剂—磷酸盐，生产低盐肉制品。可应用于水产加工品、火腿、香肠、面类、豆腐等等。TG酶在40～45℃、pH6-7的条件下，只需添加0.1-0.3%的量，即可达到明显的效果。 一、什么是TG? TG是采用现代生物工程技术研制开发出的一类用于生产新型蛋白食品的食品添加剂。TG的主要功能因子为谷氨酰胺转胺酶(EC 2.3.2.13，简称TG)，不同系列产品的主要区别在于作为辅料的蛋白质种类和数量不同。 TG产品的主要成分： 主要成分 TG TG 0.5% 1% 蛋白质99.5% 99% 二、TG具有哪些功能? TG的主要功能因子是谷氨酰胺转胺酶。这种酶广泛存在于人体、高级动物、植物和微生物中，能够催化蛋白质分子之间或之内的交联、蛋白质和氨基酸之间的连接以及蛋白质分子内谷氨酰胺残基的水解。通过这些反应，可改善各种蛋白质的功能性质，如营养价值、质地结构、口感和贮存期等。 三、TG在食品加工中有哪些用途 ? 改善食品质构。它可以通过催化蛋白质分子之间发生的交联，改善蛋白质的许多重要性能。如用该酶生产重组肉时，它不仅可将碎肉粘结在一起，还可以将各种非肉蛋白交联到肉蛋白上，明显改善肉制品的口感、风味、组织结构和营养。

谷氨酰胺转氨酶的研究进展 - 资料中心 - 生物在线

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谷氨酰胺转氨酶的研究进展 作者：陶红军， 邵虎， 黄亚东， 孔令伟， TAO Hongjun， SHAO Hu， HUANG Yadong， KONG Lingwei 作者单位：陶红军,黄亚东,TAO Hongjun,HUANG Yadong(常州红梅乳业有限公司,江苏,常州,213023)，邵虎,SHAO Hu(江苏食品职业技术学院,江苏,淮安,223003)， 孔令伟,KONG Lingwei(淮安快 鹿牛奶有限公司,江苏,淮安,223001) 刊名： 中国酿造 英文刊名：CHINA BREWING 年，卷(期)：2010(6) 参考文献(38条) 1.黄六容;何冬兰微生物谷氨酰胺酶的研究进展 2004(02) 2.王灼维;王璋土壤分离转谷氨酰胺酶生产菌株 2004(04) 3.MOTOKIM;OKIYAMA A;NONAKA M Novel transglutaminase manufacture for praparation of protein gelling compounds 1989 4.MOTOKI M;SEGURO K Transglutaminase and its use for food processing 1998 5.唐名山;王树英;陈坚Streptovcrticillinm mobaraense 谷氨酰胺转胺酶的表达、纯化和复性[期刊论文]-食品与发酵工业 2004(04) 6.鲁时瑛;岗楠迪;堵国成谷氨酰胺酶的分离纯化及酶学性质[期刊论文]-无锡轻工大学学报 2002(06) 7.崔艳华;张兰威谷氨酰胺转氨酶研究进展[期刊论文]-生物技术通报 2009(1) 8.姜燕;温其标;唐传核谷氨酰胺转移酶对食物蛋白质成膜性能的影响[期刊论文]-华南理工大学学报 2006(08) 9.丁克毅;刘军;EleanorM.Brown转谷氨酰胺酶(MTCrase)改性明胶可食件薄膜的制备[期刊论文]-食品与生物技术学报 2006(04) 10.丁克毅轻谷氨酰胺酶改性明胶高强度薄膜的制备 2006(01) 11.张春红;陈海英;车晓彦谷氨酰胺转氨酶改性谷朊粉的研究[期刊论文]-食品科学 2006(12) 12.KURAISHI C;SAKAMOTO J;YAMAZAKI K Production of restructured meat using microbial transglutaminase without salt or cooking[外文期刊] 1997(3) 13.田少君;梁华民转谷氨酰胺酶对大豆分离蛋白凝胶性的影响[期刊论文]-中国油脂 2005(08) 14.熊晓辉;王晓丽;束长丰谷氨酰胺转氨酶对内酯豆腐品质的影响[期刊论文]-食品研究与开发 2007(05) 15.田少君;梁华明转谷氨酰胺酶对大豆分离蛋白的改性研究[期刊论文]-粮油加工与食品机械 2005(06) 16.陈义华;陆兆新;尤华灰色链轮丝菌产转谷氨酰胺酶发酵条件的优化[期刊论文]-食品科学 2003(09) 17.王璋;刘新征;王亮"神舟"4号空间飞行对搭载的转谷氨酰胺酶链霉菌选育的影响[期刊论文]-航天医学与医学工程 2004(04) 18.陈国娟;张春红;刘长江谷氨酰胺酶的分离纯化及酶学性质的研究[期刊论文]-食品工业科技 2007(01) 19.LEE H G;LANIER T C;HAMANN D D Transglutaminase effects on low temperature gelation of fish protein sols[外文期刊] 1997(1) 20.ANDO H;ADACHI M;UMEDA K Purification and characteristics of a novel transglutaminase derived from microrganisms 1989 21.江波;周红霞谷氨酰胺转氨酶对大豆7S蛋白质及肌球蛋白质胶凝性质的影响[期刊论文]-无锡轻工大学学报2001(02) 22.江新业;宋钢以鱼类下脚料制备风味蛋白粉的研究[期刊论文]-中国酿造 2007(12)