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PMC3872579 | a recent systematic analysis showed that in 2011 , 314 ( 296 - 331 ) million children younger than 5 years were mildly , moderately or severely stunted and 258 ( 240 - 274 ) million were mildly , moderately or severely underweight in the developing countries .
in iran a study among 752 high school girls in sistan and baluchestan showed prevalence of 16.2% , 8.6% and 1.5% , for underweight , overweight and obesity , respectively .
the prevalence of malnutrition among elementary school aged children in tehran varied from 6% to 16% .
anthropometric study of elementary school students in shiraz revealed that 16% of them suffer from malnutrition and low body weight .
snack should have 300 - 400 kcal energy and could provide 5 - 10 g of protein / day . nowadays , school nutrition programs are running as the national programs , world - wide . national school lunch program in the united states
there are also some reports regarding school feeding programs in developing countries . in vietnam ,
school base program showed an improvement in nutrient intakes . in iran a national free food program ( nffp )
is implemented in elementary schools of deprived areas to cover all poor students . however , this program is not conducted in slums and poor areas of the big cities so many malnourished children with low socio - economic situation are not covered by nffp . although the rate of poverty in areas known as deprived is higher than other areas , many students in deprived areas are not actually poor and can afford food .
hence , nutritional value of the nffp is lower than the scientific recommended snacks for this age group .
furthermore , lack of variety of food packages has decreased the tendency of children toward nffp . on the other hand ,
the most important one is ministry of education ( moe ) of iran , which is responsible for selecting and providing the packages for targeted schools .
the ministry of health ( moh ) is supervising the health situation of students and their health needs .
welfare organizations , along with charities , have the indirect effect on nutritional status of students by financial support of their family .
provincial governors have also the role of coordinating and supervising all activities of these organizations .
parent - teacher association is a community - based institution that participates in school 's policy such as nffp .
in addition to these organizations , nutritional literacy of students , their parents and teachers , is a very important issue , which could affect nutritional status of school age children .
therefore , the present study was conducted with the aim of improving the nffp , so that by its resources all poor children will be covered even in big cities .
moreover , all food packages were replaced by nutritious and diverse packages that were accessible for non - poor children . according to the aim of this study and multiple factors that could affect the problem ,
public health advocacy has been chosen as the best strategy to deal with this issue .
therefore , the present study determines the effects of nutrition intervention in an advocacy process model on the prevalence of underweight in school aged children in the poor area of shiraz , iran .
this interventional study has been carried out between 2009 and 2010 in shiraz , iran .
this survey was approved by the research committee of shiraz university of medical sciences . in coordination with education organization of fars province
two elementary schools and one middle school in the third region of the urban area of shiraz were selected randomly . in those schools all
students ( 2897 , 7 - 13 years old ) were screened based on their body mass index ( bmi ) by nutritionists . according to convenience method all
students divided to two groups based on their economic situation ; family revenue and head of household 's job and nutrition situation ; the first group were poor and malnourished students and the other group were well nourished or well - off students .
for this report , the children 's height and weight were entered into center for disease control and prevention ( cdc ) to calculate bmi and bmi - for - age z - scores based on cdc for diseases control and prevention and growth standards .
the significance of the difference between proportions was calculated using two - tailed z - tests for independent proportions . for implementing the interventions ,
the advocacy process model weight was to the nearest 0.1 kg on a balance scale ( model # seca scale ) .
standing height was measured to the nearest 0.1 cm with a wall - mounted stadiometer .
advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders .
the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities . situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts .
the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed .
accordingly , three sub - groups were established : research and evaluation , education and justification and executive group . designing the strategies :
three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking . performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge .
education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) .
accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style .
it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children .
after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them .
for educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support .
healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on .
nutritional intervention : the snack basket of the students was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron .
low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves .
research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions .
the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean .
each student that was malnourished and poor has been taken into account for free food and nutritious snacks .
demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire .
this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province .
statistical analyses were performed using the statistical package for the social sciences ( spss ) software , version 17.0 ( spss inc . ,
the results are expressed as mean sd and proportions as appropriated . in order to determine the effective variables on the malnutrition status
paired t test was used to compare the end values with baseline ones in each group .
in this project , the who z - score cut - offs used were as follow : using bmi - for - age z - scores ; overweight : > + 1 sd , i.e. , z - score > 1 ( equivalent to bmi 25 kg / m ) , obesity : > + 2 sd ( equivalent to bmi 30 kg / m ) , thinness : < 2 sd and severe thinness : < 3 sd .
this interventional study has been carried out between 2009 and 2010 in shiraz , iran .
this survey was approved by the research committee of shiraz university of medical sciences . in coordination with education organization of fars province
two elementary schools and one middle school in the third region of the urban area of shiraz were selected randomly . in those schools all
students ( 2897 , 7 - 13 years old ) were screened based on their body mass index ( bmi ) by nutritionists . according to convenience method all
students divided to two groups based on their economic situation ; family revenue and head of household 's job and nutrition situation ; the first group were poor and malnourished students and the other group were well nourished or well - off students .
for this report , the children 's height and weight were entered into center for disease control and prevention ( cdc ) to calculate bmi and bmi - for - age z - scores based on cdc for diseases control and prevention and growth standards .
the significance of the difference between proportions was calculated using two - tailed z - tests for independent proportions . for implementing the interventions ,
weight was to the nearest 0.1 kg on a balance scale ( model # seca scale ) .
standing height was measured to the nearest 0.1 cm with a wall - mounted stadiometer .
advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders .
the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities . situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts .
the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed .
accordingly , three sub - groups were established : research and evaluation , education and justification and executive group . designing the strategies :
three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking . performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge . duration of intervention was 6 months .
education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) .
accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . obviously , student 's families had remarkable effect on children 's food habit .
it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children .
after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them .
educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support .
healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on .
nutritional intervention : the snack basket of the students was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron .
low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves .
research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions .
the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean .
each student that was malnourished and poor has been taken into account for free food and nutritious snacks .
demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire .
this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province .
advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders .
the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities .
situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts .
the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed .
accordingly , three sub - groups were established : research and evaluation , education and justification and executive group .
designing the strategies : three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking .
performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge .
education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) .
accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . obviously , student 's families had remarkable effect on children 's food habit .
it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children .
after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them .
educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support .
healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on . nutritional intervention : the snack basket of the students
was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron .
low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves .
research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions .
the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean .
each student that was malnourished and poor has been taken into account for free food and nutritious snacks .
demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire .
this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province .
statistical analyses were performed using the statistical package for the social sciences ( spss ) software , version 17.0 ( spss inc . , chicago , il , usa ) .
the results are expressed as mean sd and proportions as appropriated . in order to determine the effective variables on the malnutrition status
paired t test was used to compare the end values with baseline ones in each group .
two - sided p < 0.05 was considered to be statistically significant . in this project ,
the who z - score cut - offs used were as follow : using bmi - for - age z - scores ; overweight : > + 1 sd , i.e. , z - score > 1 ( equivalent to bmi 25 kg / m ) , obesity : > + 2 sd ( equivalent to bmi 30
kg / m ) , thinness : < 2 sd and severe thinness : < 3 sd .
study population contains 2897 children ; 70.8% were primary school students and 29.2% were secondary school students .
2336 ( 80.5% ) out of total students were well - off and 561 children ( 19.5% ) were indigent .
19.5% of subjects were in case group ( n = 561 ) and 80.5% were in the control group ( n = 2336 ) .
the mean of age in welfare group was 10.0 2.3 and 10.5 2.5 in non - welfare group .
demographic characteristics of school aged children in shiraz , iran table 2 shows the frequency of subjects in different categories of bmi for age in non - welfare and welfare groups of school aged children separately among boys and girls before and after a nutrition intervention based on advocacy process model in shiraz , iran .
the frequency of subjects with bmi lower than < 2 sd decreased significantly after intervention among non - welfare girls ( p < 0.01 ) .
however , there were no significant decreases in the frequency of subjects with bmi lower than < 2 sd boys .
when we assess the effect of intervention in total population without separating by sex groups , we found no significant change in this population [ table 3 ] .
bmi for age for iranian students aged 7 - 14 years based on gender according to who growth standards 2007 bmi for age for iranian students aged 7 - 14 years according to who growth standards 2007 in non - welfare and welfare groups of total population table 4 has shown the prevalence of normal bmi , mild , moderate and severe malnutrition in non - welfare and welfare groups of school aged children separately among boys and girls before and after a nutrition intervention based on advocacy process model . according to this table
there were no significant differences in the prevalence of mild , moderate and severe malnutrition among girls and boys .
table 4 also shows the mean of all anthropometric indices changed significantly after intervention both among girls and boys .
the pre- and post - test education assessment in both groups showed that the student 's average knowledge score has been significantly increased from 12.5 3.2 to 16.8 4.3 ( p < 0.0001 ) .
bmi , height and weight in non - welfare and welfare groups of school aged children separately in males and females before and after a nutrition intervention based on advocacy process model in shiraz , iran according to study 's finding the odds ratio ( or ) of sever thinness and thinness in non - welfare compared with welfare is 3.5 ( or = 3.5 , confidence interval [ ci ] = 2.5 - 3.9 , p < 0.001 ) .
furthermore , the finding showed or of overweight and obesity in welfare compared to non - welfare is 19.3 ( or = 19.3 , ci = 2.5 - 3.9 , p = 0.04 ) .
the result of this community intervention study revealed that nutrition intervention based on advocacy program had been successful to reduce the prevalence of underweight among poor girls .
this study shows determinant factor of nutritional status of school age children was their socio - economic level . according to our knowledge ,
this is the first study , which determines the effect of a community intervention based on advocacy process on the malnutrition indices in a big city ( shiraz ) in iran .
the other program in iran ( nffp ) is specified to deprived area and is not conducted in big cities .
allocating millions of dollars to nffp by government , selecting the malnourished students through an active screening system at primary and middle schools , paying attention of policy makers to student 's nutrition have provided the opportunity to combat the problem . however , negligence of under - poverty line , providing poor snacks in terms of nutritional value and lack of variety are the main defects of this program .
advocacy by definition is a blending of science , ethics and politics for comprehensive approaching health issues . by using advocacy program in california among the high school students for improving their nutrition and physical activity
angeles unified school district participants emphasized on nutrition classes for families as well as students in addition to other interventions . in the present study
another study revealed that evaluability assessment gave stakeholders the opportunity to reflect on the project and its implementation issues .
it seems that in iran , free food program among the students not only is needed in deprived areas , but also it should be performed in big cities such as shiraz . at baseline ,
no significant difference was founded among wealthy students between the pre- and post - nutritional status intervention .
in contrast , the numbers of students who have malnutrition decreased from 44% to 39.4% , which was identified as a significant among impecunious girls students .
there was also a significant increase in the proportion of children with bmi that was normal for age ( 2 to + 1 sd ) most of the published community interventions showed better results among females compared with males .
this difference in the impact of nutritional interventions between male and female might be related to the different age of puberty in the female population compared to the male population . in the age range of the present study female
although , there is no nffp in big cities of iran , there are some programs for improving the nutritional status such as providing free milk in schools .
a recent publication has shown that school feeding programs focus on milk supplementation had beneficial effects on the physical function and school performances specifically among girls in iran .
the results of the mentioned study showed an improvement in the weight of children , psychological test 's scores and the grade - point average following this school feeding program .
the intervention in the present study had focused on the snack intake in the school time .
there are some reports regarding the nutrition transition in iran , which shows the importance of nutrition intervention to provide more healthy eating dietary habits among welfare groups of adolescents .
hence , nutrition intervention especially in the form of nutrition education is needed in big cities and among welfare children and adolescents . although a study among iranian adolescents showed that dietary behavior of adolescents does not accord to their knowledge , which emphasize on the necessity of community intervention programs . a recent study regarding the major dietary pattern among iranian children showed the presence of four major dietary patterns , in which fast food pattern and sweet pattern as two major dietary patterns can be mentioned among iranian children . in advocacy program audience 's analysis
accordingly , one of the prominent strategies in this study was working with media and was meeting with parent - teacher association that both of them were secondary target audiences
. we also took into account policy makers in different levels , from national to local as primary audiences .
advocacy team had several meetings with management and planning organization at national level and education organization of the fars province as well as principal of the targeted schools .
providing nutritious snacks need contribution of private sector such as food industries or factories , but their benefits should be warranted .
another choice was community involvement ; which can be achieved by female health volunteers who are working with the health system .
advocacy team by using the support of charities and female health volunteers could establish a local factory that produced student 's snacks based on the new definition . however , there are some challenges on the way of expanding this program .
mass production of the proposed snacks according to different desires and cultures and getting involvement of food industries with respect to marketing issues is one of those challenges .
moreover , providing a supportive environment in order to change the food habits of the students and their parents among the wide range of the population require a sustainable and continuous inter - sector collaboration .
although in a limited number of schools , in our study , interventions and advocacy program was successful , expanding this model to another areas around the country depends on convincing the policy makers at national level . in this
regard , advocacy team should prepare evidenced based profile and transitional planning to convince the policy makers for improving the rule and regulation of nffp .
the same as this study in other studies have also emphasized that there must be efforts to strengthen the capacity within the schools to deal with the nutritional problems either overweight , obesity or malnutrition by using of educational and nutritional intervention .
assessing the dietary adherence is very important in nutrition intervention among population . as this population was children and adolescents we had a limitation in the blood sample collection to assess the subject 's dietary adherence .
furthermore , this intervention was only focused on the intake of snack in school time and we did not have comprehensive information on the dietary intake of children and adolescents after school all over the day .
the investigators propose further investigation in different areas of the country based on socio - cultural differences in order to make necessary modification and adapt this model to other areas .
regarding the nutritional needs of the school age children , provision of a good platform for implementing and expanding this efficient model to the whole country based upon the socio - economic situation of each region is advisable to the moh and the moe .
community nutrition intervention based on the advocacy process model is effective on reducing the prevalence of underweight specifically among female school aged children . | background : the present study was carried out to assess the effects of community nutrition intervention based on advocacy approach on malnutrition status among school - aged children in shiraz , iran.materials and methods : this case - control nutritional intervention has been done between 2008 and 2009 on 2897 primary and secondary school boys and girls ( 7 - 13 years old ) based on advocacy approach in shiraz , iran .
the project provided nutritious snacks in public schools over a 2-year period along with advocacy oriented actions in order to implement and promote nutritional intervention . for evaluation of effectiveness of the intervention growth monitoring indices of pre- and post - intervention were statistically compared.results:the frequency of subjects with body mass index lower than 5% decreased significantly after intervention among girls ( p = 0.02 ) .
however , there were no significant changes among boys or total population .
the mean of all anthropometric indices changed significantly after intervention both among girls and boys as well as in total population .
the pre- and post - test education assessment in both groups showed that the student 's average knowledge score has been significantly increased from 12.5 3.2 to 16.8 4.3 ( p < 0.0001).conclusion : this study demonstrates the potential success and scalability of school feeding programs in iran .
community nutrition intervention based on the advocacy process model is effective on reducing the prevalence of underweight specifically among female school aged children . | INTRODUCTION
MATERIALS AND METHODS
Participants
Instruments
Procedure
First step
Second step
Third step
Forth step
Interventions
Fifth step (assessment)
Data analysis
RESULTS
DISCUSSION
CONCLUSION |
PMC5330001 | tardive dystonia ( td ) , a rarer side effect after longer exposure to antipsychotics , is characterized by local or general , sustained , involuntary contraction of a muscle or muscle group , with twisting movements , generally slow , which may affect the limbs , trunk , neck , or face .
td has been shown to develop in about 3% of patients who have had long - term exposure to antipsychotics .
. the low risk of td for atypical antipsychotics is thought to result from their weak affinity for dopamine receptors . compared with typical ,
atypical antipsychotic agents have a greater affinity for serotonin 5-ht2a than dopamine d2 receptors , with a low propensity to induce td . among this olanzapine
is thought to have preferential action at mesolimbic over nigrostriatal dopaminergic pathways and is , therefore , associated with a very low incidence of extrapyramidal symptom ( eps ) .
furthermore , a retrospective analysis of controlled multicentric trials suggested that olanzapine also improves preexisting symptoms of tardive movements .
we report a case of 20-year - old male , belonging to lower socioeconomic class , educated up to 2 standard , presented with severe unilateral dystonic left sided neck movements [ figure 1 ] .
careful history exploration revealed he was taking risperidone 2 mg irregularly for 2 months and then olanzapine 5 mg for another 4 months .
picture of neck dystonia of patient at 19 years , the patient presented with occasional anger outbursts , getting provoked on small matters and beating family members , running away from home , screaming episodes occasionally , fearfulness , sleep disturbance for 2 days ; which was precipitated after fever . according to the mother
, one friend might have threatened / made fun of him actually and after that patient stopped going out of house , and displayed above mentioned symptoms .
this was interpreted as psychosis with persecutory ideas , and he was treated with risperidone 2 mg / day for 2 months and then with olanzapine 5 mg / day for 4 months . in last two follow - ups patient did not present himself , and mother reported unusual neck movements , which were taken as a part of his overall psychopathology and not taken seriously , slight intermittent neck movements reported were missed as part of adolescent behavior problems mimicking some hero in movies . as neck dystonia increased , the patient had a severe disability as patient had to keep his hands behind his head for the support .
the movement would decrease when the patient was lying down and was absent during sleep .
he even stopped taking food due to severe neck movements making chewing and swallowing difficult .
his birth and early developmental milestones were normal . during 210 years of age patient was inattentive and mildly hyperactive .
other siblings were educated with master 's degree , and patient was also sent to school , but due to inattention and restlessness , he did not pass 2 standard after three attempts .
he left the schooling . with average executive functioning and life skills , he worked as an unskilled laborer in the neighborhood shops as a helping hand .
, he was found to be getting over familiar , cheerful , moody , and short tempered .
sometimes , the patient had inappropriate social judgment ; for which his friends made fun of him , and teased him . on mental status assessment ,
routine investigations , thyroid function tests , electroencephalogram , fundus examination , cervical x - ray , magnetic resonance imaging brain were normal . after consulting neurophysician , wilson 's disease and other secondary causes of dystonia were ruled out .
the patient was treated with clonazepam 1 mg total dissolved solid ( tds ) , tetrabenazine 25 mg tds , trihexiphenidyl 2 mg bipolar disorder ( bd ) . after 2 months , there was some improvement of around 30% .
baclofen 10 mg was added ; increased up to 20 mg , trihexyphenidyl reduced to 2 mg . with little improvement after 4 months of treatment for dystonia , levodopa + carbidopa ( 100 + 25 ) was added by neurophysician and increased up to tablet tds and baclofen omitted .
after 12 months of treatment , patient has improved around 90% with tetrabenazine 75 mg , levodopa + carbidopa ( 100 + 25 ) - tablet bd , and clonazepam 1 mg bd .
earlier case reports reported td developing with high - dose atypical antipsychotics such as olanzapine 20 mg or aripiprazole 15 mg with longer duration of exposure of around 1215 months in established psychiatric illness like schizophrenia or any other psychotic illness .
eps in general and tardive dyskinesia , in particular , have been extensively studied in schizophrenia . even though a number of studies suggest that bipolar patients experience higher rates of eps ( parkinsonism , dystonia , akathisia ) and td compared to patients with a diagnosis of schizophrenia , research within the bd population has been limited .
the risk is found to be 3 to 5 times higher in elderly patients compared to young patients .
in addition to age , the risk is directly proportional to : female gender , daily and total dose of the antipsychotic drug , presence of mood disorder , the use of anticholinergics with neuroleptics , previous physical therapies ( electroconvulsive therapy ) , the presence of other physical illness such as diabetes or an organic disorder , younger age of exposure , and the presence of extrapyramidal symptoms early in treatment .
this patient 's severe dystonic neck movements developed within short period of 6 months of exposure to atypical antipsychotics risperidone 2 mg and then olanzapine 5 mg only , which can cause minimal extrapyramidal side effects . in this case ,
risk factors for developing serious disabling td were neuroleptic exposure , borderline intellectual functioning , externalizing behavior , probable misdiagnosis , and overlooking early indicators of side effects .
this case highlights dangers of casually prescribing low dose second generation antipsychotics in patient with hyperthymic temperament and borderline intellectual functioning with vague short lasting presenting complaints ; probably misdiagnosed as psychosis ; leading to such severe adverse effects because patients with organic brain damage are more prone to develop adverse effects like td .
thus , judicious use of antipsychotics , with detailed and frequent assessments is important , and emergent stereotyped behavior or unexplained movements must be examined carefully and taken seriously .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . | tardive dystonia ( td ) is a serious side effect of antipsychotic medications , more with typical antipsychotics , that is potentially irreversible in affected patients .
studies show that newer atypical antipsychotics have a lower risk of td . as a result , many clinicians may have developed a false sense of security when prescribing these medications .
we report a case of 20-year - old male with hyperthymic temperament and borderline intellectual functioning , who developed severe td after low dose short duration exposure to atypical antipsychotic risperidone and then olanzapine .
the goal of this paper is to alert the reader to be judicious and cautious before using casual low dose second generation antipsychotics in patient with no core psychotic features , hyperthymic temperament , or borderline intellectual functioning suggestive of organic brain damage , who are more prone to develop adverse effects such as td and monitor the onset of td in patients taking atypical antipsychotics . | INTRODUCTION
CASE REPORT
DISCUSSION
Declaration of patient consent
Financial support and sponsorship
Conflicts of interest |
PMC4301082 | the guanine heterocyclic
ring is rich in chemical reactivity toward
oxidants and adduct - forming species . in dna , 2-deoxyguanosine
( dg , mass = m ) is the chief site for base oxidation due
to its low redox potential leading to many products that are characterized
by their mass signatures .
the principal products
characterized with one - electron oxidants in aqueous solutions can
be grouped based on the site of reaction on the radical intermediate
resulting from one - electron oxidation of dg .
the 2-deoxyribonucleoside products arising from initial
reactivity at c5 of dg include a four - electron oxidation
product , an imidazolone ( diz , m-39 ) that hydrolyzes to
an oxazolone ( dz , m-21 ) , and
the two - electron oxidation product 5-carboxamido-5-formamido-2-iminohydantoin
( d2ih , m+34 ; scheme 1 ) .
when the initial reaction occurs at c8 of dg , either
2,6-diamino-4-hydroxy-5-formamidopyrimidine ( fapy - dg ,
m + 18 ) is observed under reducing conditions or 8-oxo-7,8-dihydroguanine
( dog , m+16 ) is observed under oxidizing conditions , in
which the latter compound is a key marker for monitoring oxidative
stress in cells ( scheme 1 ) .
the
two - electron oxidation product dog is stable but is highly
susceptible to further oxidation leading to two hydantoin compounds .
the yield for the thermodynamically preferred product spiroiminodihydantoin
( dsp , m + 32 ) is greatest in nucleoside reactions at
ph > 6 or in unencumbered reaction contexts ( i.e. , single - stranded
dna or g - quadruplexes ) , while the yield of 5-guanidinohydantoin ( dgh , m + 6 ) is greatest in nucleoside reactions at ph <
6 or in sterically demanding duplex contexts ( scheme 1 ) .
the hydantoins are also readily formed from direct four - electron
oxidation with o2 .
this list of products represents those that are consistently observed
from many oxidant systems conducted by several laboratories ; however ,
other compounds have been reported in lower yield . in the cellular context
, oxidation reactions
with dg can have other nucleophilic participants such
as amines or phenols .
in most cases , when amines or phenols participate in the reaction ,
products
are observed with similar backbone structures as characterized
with water serving as the nucleophile .
for example , when dg is oxidized in the presence of lysine , products retaining the dg heterocycle and those with a core similar to dgh or dsp have been observed .
the adducts characterized by
our laboratory and others show lysine competing with water for sites
of covalent bond formation , where a single lysine is observed at c8
analogous to dog , at either c5 or c8 of a spirocyclic
core similar to dsp , and last a bis - adduct at c5 and
c8 with a spirocyclic ring structure has been identified ( figure 1 ) .
the lysine
adducts are all stable allowing their quantification and characterization .
as a last example
, the polyamine spermine has been adducted to dg and dog under oxidative conditions . in the dg oxidation studies , a spermine adduct at c8
is observed
retaining the dg heterocyclic core , while dog oxidations yield an adduct at c5
that does not undergo acyl migration to create a spirocycle , but instead
the polyamine can generate an unstable hemiaminal intermediate that
decomposes leaving a ribosylurea lesion at the adduct formation site .
moreover , the facile formation of spermine adducts
to dog oxidation intermediates has been harnessed for
quantification of dog from dna samples .
in contrast to amines , phenols ( i.e. , tyrosine ) are more
redox active than amines and better able to compete with dg for oxidant during reactions .
the products observed when phenols
participate with dg during oxidation have more variability
in their structures compared to amine adducts .
these model studies aid in understanding
the chemical nature of dna protein cross - links that are detrimental
to cellular processes . in the current work ,
oxidations that allowed dg to
react with nh3 were conducted while the full product and
diastereomer distributions were monitored .
after careful characterization
of the nucleoside reactions , similar oxidations were conducted in
the single - stranded and duplex oligodeoxynucleotide ( odn ) contexts .
the purified ammonia adducts were also studied with respect to their
decomposition pathways leading to end products that have not been
previously determined .
lastly , polymerase insertion studies were conducted
to test the hypothesis that amine adducts might have altered base - pairing
preferences ; every substitution of an oxo group on a base for an amino
group converts a hydrogen bond acceptor into a potential hydrogen
bond donor .
these results provide a fundamental understanding of the
stability and base - pairing properties of amine adducts to dg that result during oxidation , which may occur in vivo due to the
high concentration of nucleophilic amines in the vicinity of the genome .
adducts
observed when dg and lysine are allowed to
react in the presence of oxidant .
the unencumbered nucleoside dg ( 1 mm ) was chosen for
the initial oxidation reactions where nh4cl ( 20 mm ) provided
the source of nucleophilic nh3 .
all reactions were conducted
with 75 mm napi buffer at ph 7.4 at 22 c .
the oxidants
chosen include the photooxidants riboflavin and rose bengal and the
one - electron oxidant na2ircl6 .
reactions were
conducted in triplicate to achieve 70% conversion to products
in each reaction , and the products ( scheme 2 ) were analyzed by a dual hplc method . the first round of hplc analysis
utilized a reversed - phase column to identify dog ( m+16 )
and 8-amino - dg ( m+15 ) , while all other products eluted
in the void volume .
the void volume from the previous run was collected
and analyzed on a hypercarb hplc column that allowed analysis of the
hydantoins , ammonia adducts , and dz .
however , diz hydrolyzes
to dz that was detected on the hypercarb column ; thus ,
formation of diz is inferred from quantification of dz .
moreover , the hypercarb hplc column also allowed separation
and quantification of all spirocyclic diastereomeric pairs of products .
the dgh diastereomers are interconvertible , and thus , their diastereomer ratios are not
reported ( see the supporting information for complete experimental details ) .
lastly , a test reaction with
na2ircl6 was conducted in which half was directly
analyzed by the hypercarb hplc column and the other half was analyzed
by the dual hplc method outlined above .
the product distributions
observed from these comparative studies were within 3% of
one another .
the photooxidant riboflavin led to the largest number
of different
products and was used to obtain suitable amounts of material for characterization .
esi - ms ( figure s1 , supporting information ) , and the adducts observed that did not involve participation of
nh3 during product formation included dz ( m
- 21 ) , dog ( m + 16 ) , dgh ( m + 6 ) , and dsp ( m + 32 , scheme 2 ) .
confirmation
of their structures was achieved by esi - ms / ms fragmentation
of the free bases while monitoring the daughter fragments .
the esi - ms / ms experiments were conducted on hplc purified nucleosides
in which the n - glycosyl bond was cleaved in the ionization
source to liberate free bases that were further fragmented in the
cid chamber to generate the daughter fragments used in identification
of the structures .
the ms / ms fragmentation spectra were compared to
literature values for the two dsp diastereomers ( figure 2 ) and dz to further confirm their structural
assignments ( figures s2 and s3 , supporting information ) .
the structure for dsp has been further established
by x - ray crystallography and nmr .
ammonia adducts observed included two pairs
of chromatographic peaks for the diastereomers of spirodi(iminohydantoin)-2-deoxyribonucleosides
( dsi ) , whose names are based on the site of nh3 attachment to guanine , thus 5-dsi ( m + 31 ) and 8-dsi ( m + 31 ) ( scheme 2 , see reference
cited for the correct dsi iupac nomenclature ) . to confirm the identities of the 5- and 8-dsi constitutional isomers and their respective
diastereomers , esi - ms / ms of the free bases were conducted
( figure 2 ) .
comparison of the esi - ms / ms spectrum for 5-dsi with dsp gave
a pair of peaks for 5-dsi that established c5 as the
site of nh3 attachment ( m / z [ m + h ] = 140 and 96 , figure 2 ) .
as for 8-dsi , comparison of its esi - ms / ms
spectrum with those obtained from dsp and 5-dsi identified new masses that are best explained by nh3 attachment
at c8 ( m / z [ m + h ] =
141 and 97 , figure 2 ) . on the basis of the
proposed mechanism ( scheme 2 ) , a product that
includes bis - addition of nh3 at c5 and c8 of an oxidized
guanine to yield a spirocycle ( 5,8-dsi , m+30 ) is possible ; however , this adduct was not observed
for reasons that will be elaborated on below .
additionally , nh3 adducts with a dgh core were not observed most
likely due to the fact that the reactions were performed at ph 7.4
where yields of dgh are minimal .
lastly , the yields of dog ( m + 16 ) and 8-amino - dg ( m + 15 ) were very
low ( < 1% ) as determined by lc - esi - ms ( figure s1 , supporting information ) ; hence , their quantities
are not reported .
the nucleosides dog and c8 amine - adducted dg have redox potentials that are 600 mv below the
parent nucleoside dg causing them to be much more susceptible
to further oxidation .
further , one - electron oxidized dog was observed to have an even lower redox potential than
the parent compound dog , which means that once oxidation of dog , and likely 8-amino - dg , occurs product formation is inevitable due to
the 70% conversion to product that yielded the spirocyclic
compounds dsp , 5-dsi , or 8-dsi .
esi - ms / ms spectra for dsp ( top ) , 5-dsi ( middle ) , and 8-dsi ( bottom ) .
the data
provided was collected on the first eluting diastereomer of each spirocycle
from a hypercarb hplc column .
data for the second eluting diastereomer
for dsp , 5-dsi , and 8-dsi can
be found in the supporting information ( figures
s2 , s4 , and s5 ) .
the heavy lines represent the fragment observed and
the thin lines represent the portion of the molecule lost upon fragmentation .
relative product distributions
observed with each oxidant system
were determined by integration of the hplc peak areas measured at
240 nm followed by normalization via each molecule s unique
extinction coefficient ( 240 nm ) .
extinction coefficients for 5-dsi and 8-dsi are not known but were determined from experiments that
are reported below . in the riboflavin - mediated oxidations ,
this result was anticipated because riboflavin is a type i photooxidant
and effects oxidation by electron transfer from dg yielding
o2 under aerobic reaction conditions .
oxidation of dg by one - electron
and proton transfer yields an intermediate radical ( dg ) that only couples with o2 to initially yield diz that
is prone to hydration leading to dz ( scheme 2 ) , the species quantified .
the adducts
derived from the nucleophile trapping of electrophilic dg oxidation intermediates show nh3 participation through
the products 5-dsi ( 29% ) that was the highest yielding
nh3 adduct followed by nearly 3-fold less 8-dsi ( 11% ) .
the participation of h2o as the trapping nucleophile
was identified by the lower yields of dsp ( 11% ) and dgh ( 4% ) .
the nucleophilicity of nh3 is much greater
than h2o ; therefore , as expected , adducts resulting from
nh3 participation dominated over those derived from h2o .
relative product distributions observed when dg reacted
with nucleophilic h2o or nh3 in oxidation reactions .
the oxidants include the photooxidants riboflavin and rose bengal ,
as well as na2ircl6 .
reactions were conducted
with 1 mm dg , 20 mm nh4cl , in 75 mm napi buffer ( ph 7.4 ) at 22 c .
( 1 ) photoactivation of riboflavin
( 200 m ) was achieved with 350 nm light for 3 h , ( 2 ) photoactivation
of rose bengal ( 100 m ) was achieved with 350 nm light for 3
h , and ( 3 ) oxidation with na2ircl6 ( 10 mm ) was
achieved by bolus addition of the salt .
results represent the average
of triplicate trials and the error is 10% of each reported
value .
the second oxidant studied was
rose bengal , a type ii photooxidant
( o2 ) that furnished 5-dsi as the
major product ( 62% ) followed by a 3-fold lower amount of dsp ( 31% ) , and low yields of dz ( 5% ) and dgh ( 2% ) were observed to complete the mass balance ( figure 3 ) . as expected , this oxidant did not yield 8-dsi based on its mechanism of oxidation .
oxidation of dg by o2 proceeds by 4 + 2 cycloaddition
to the imidazole ring , followed by ring opening to yield 8-hoo - g that eliminates water giving the proposed electrophile dog .
next , dog is trapped by nucleophiles at
c5 leading to dsp with h2o or 5-dsi with nh3 ( scheme 3 ) .
support for
exclusive nucleophilic attack at c5 under o2 oxidations was derived from h2o studies
followed by mapping the labeled site by esi - ms / ms ; the current observation that the only nh3 adducts were the 5-dsi diastereomers further
support this previously proposed mechanism , and further confirms our
structural assignments for these peaks .
comparisons of the product
distributions from the photooxidants riboflavin and rose bengal show
a dramatic difference with respect to the major product observed ( figure 3 ) . in the riboflavin oxidation ,
dz was
the major product while in the rose bengal oxidation , 5-dsi was the major product , and these observations can be ascribed to
each oxidant s unique mechanism of oxidation ( schemes 2 and 3 ) .
product distributions from this oxidant
included
nearly equivalent distributions of dsp ( 50% ) and 5-dsi ( 42% ) with the mass balance completed by dgh ( 8% , figure 3 ) . in this oxidation reaction
,
the distribution did not yield nh3 adducts as the major
products , which was unexpected on the basis of nh3 being
the better nucleophile compared to h2o .
comparison
of the product distributions observed with the one - electron
oxidants riboflavin and na2ircl6 identified
both nh3- and h2o - adducted compounds .
the current
results differ from those previously reported by our laboratory when
lysine was adducted to dg in analogous oxidation reactions . previously , in riboflavin oxidations spirodihydantoins
with lysine at c8 were 2-fold greater than those with lysine at c5
( figure 1 ) , and in na2ircl6 oxidations a c8 lysine adduct was observed .
in contrast to these
results , riboflavin oxidations in the presence of nh4cl
gave more c5 adducts ( 29% ) than c8 adducts ( 11% ) , and na2ircl6 oxidations did not yield detectable amounts of c8
adducts .
we propose the difference in the current ammonia results
compared to the lysine data can be attributed to a difference in the
mechanism of product formation for the c8 adduct in the nucleoside
context .
the former work from our laboratory proposed that c8 amine
adducts result from oxidation of the amine to the aminyl radical that
adds at the c8 carbon of dg followed by oxidation leading
to product formation ( scheme 4 ) . in nucleoside dg , this proposed
mechanism best supports the observation of ammonia adducts at c8 ,
because upon one - electron oxidation of dg the initial
radical cation ( dg )
formed is very acidic ( pka 3.9 ) and rapidly deprotonates to the neutral radical
( dg ) that is not susceptible
to nucleophilic attack . thus , dg reacts with o2 yielding diz / dz and not with amines or
h2o .
formation of amine adducts at c8 must result
from a difference in the amine reactivity .
the key difference between
lysine and ammonia resides in their standard reduction potentials .
in general , primary amines ( 1.0 v vs nhe , ph 10 ) have a lower redox potential than ammonia ( > 1.3 v vs
nhe ,
ph 9 ) ; this trend should scale down to
ph 7 , in which the oxidations were conducted .
thus , oxidation of lysine
to an aminyl radical that adds to c8 of dg is possible
with na2ircl6 ( 0.9 v vs nhe , ph 7 ) and riboflavin
( 1.7 v vs nhe , ph 7 ) ; in contrast , the
analogous reaction does not readily occur for nh3 because
at ph 7 dg is the dominant site of oxidation due to its
lower redox potential leading to products other than 8-dsi . in summary ,
one - electron oxidant driven oxidations of dg in the presence of nh3 lead to spirocyclic adducts at
c5 and c8 that are in competition with h2o adducts of the
same core structure . as the next step , we set out to determine the decomposition
products
and pathways through which the ammonia adducts proceed .
hplc was used
to provide diastereomerically pure 5-dsi and 8-dsi samples that were subjected to conditions of ph 3 ( 0.1% formic acid )
or ph 10 ( 20 mm napi ) at 22 c for 30 min or 10 h.
not only could the decomposition products be determined , but having
diastereomerically pure starting material also allowed us to probe
the mechanism of decomposition , specifically for 5-dsi .
first , both 5- and 8-dsi were stable
at ph 10 during this time frame , while 5-dsi readily
hydrolyzed to dsp at ph 3 in 30 min and 8-dsi hydrolyzed to dsp after 10 h. for 5-dsi , deamination of the amine group can occur by two possible mechanisms :
( 1 ) a retro - acyl migration can occur followed by loss of nh3 to yield an electrophilic intermediate ( dog ) susceptible to h2o attack followed by
a second acyl migration back to dsp or ( 2 ) acid - catalyzed
deamination can occur directly to furnish dsp ( scheme 5 ) .
if the retro - acyl migration proceeds on the diastereomerically
pure sample , it is expected that a mixture of dsp diastereomers
would be observed , and if direct deamination occurs , then only one dsp diastereomer would be predicted . upon incubation of one
diastereomer of 5-dsi at ph 3 ,
only one diastereomer
of dsp was observed , supporting the direct deamination
mechanism .
moreover , the early eluting 5- and 8-dsi nh3 adducts decomposed to the early eluting dsp isomer
and the late eluting adducts deaminated to the later eluting dsp isomer ( figure s6 , supporting information ) .
these results aid in establishing the absolute configuration of
the 5-dsi and 8-dsi diastereomers that will
be discussed below .
moreover , these results also allude to an explanation
as to why the bis - ammonium adduct was not observed in any of the reactions .
based on these data , the bis - ammonium adduct , if formed , could rapidly
deaminate to yield either 5-dsi or 8-dsi , although this hypothesis could not be further validated .
utilization of the hypercarb
hplc column for analyzing the product
distributions of these spirocycles allowed determination of the diastereomer
ratios .
it has previously been determined that ( r)-dsp elutes first from this
column and ( s)-dsp elutes second .
the absolute stereochemistry for the diastereomers
of the ammonia adducts had not yet been determined .
the results of
these studies identified ( r)-dsp and ( s)-dsp yields to be nearly equal . also , for the 5-dsi and 8-dsi ammonia adducts nearly equal
yields for the diastereomers were observed . on the basis of these
results , the defining point of the reaction to determine product stereochemistry
must not be very sterically impeded in order to lead to such a small
diastereomer preference . as stated above , the diastereomerically
pure ammonia adduct samples
were determined to decompose to give a single diastereomer of dsp ; therefore , on the basis of hplc elution order , the absolute
configuration for the diastereomers of the two dsi constitutional
isomers can be determined .
the first - eluting 5-dsi and 8-dsi diastereomers decomposed to the first dsp diastereomer ( r ) , and the late - eluting diastereomer
decomposed to the late - eluting dsp diastereomer ( s ) ; therefore , for 5-dsi the r diastereomer elutes first and the s diastereomer
elutes second , because the r and s assignments are the same between dsp and 5-dsi .
in contrast , the r and s assignments
for the 8-dsi diastereomers are opposite those of dsp ; thus , ( s)-8-dsi elutes first and ( r)-8-dsi elutes second and from the hypercarb
hplc column ( figure 4 ) .
these examples provide
a fascinating case outlining how the movement of ring substituents
affects the r and s stereochemical
assignments .
assignment of absolute configurations for the diastereomers
of dsp , 5-dsi , and 8-dsi based
on their
elution profile from a hypercarb hplc column and their ecd spectra .
the r and s assignments for the diastereomers
of 8-dsi are the opposite of those for the dsp and 5-dsi isomers with the same geometric configuration
of the spirocyclic ring , due to a change in the cahn ingold
the diastereotopic ammonia
adducts were then probed by electronic
circular dichroism spectroscopy ( ecd ) .
previously , ecd was used in
tandem with vibrational circular dichrosim spectroscopy , nmr , and
x - ray crystallography to establish the absolute configuration for dsp .
the ecd spectra for ( r)- and ( s)-dsp isomers gave three lobes that were mirror images
of one another , as expected ( figure 4 ) .
critical
for assigning the absolute configuration for dsp was
the low energy lobe , in which the s isomer gave a
positive rotation at 258 nm , and the r isomer gave
a negative rotation at 259 nm . in comparison
to these results ,
the 5-dsi diastereomers gave ecd spectra
that were different than dsp but showed a similarity
in the low energy lobe ( figure 4 ) .
for ( r)-5-dsp , the
lobe at 258 nm gave a negative rotation and ( s)-5-dsi gave a positive rotation
at 258 nm .
the higher energy lobes observed in the
ecd for the 5-dsi diastereomers were different than those
measured for dsp ( figure 4 ) .
as
a last comparison , the 8-dsi diastereomer ecd spectra
were compared to those recorded for dsp ( note : 8-dsi and dsp isomers with the same geometric
configuration of the spirocyclic ring give opposite r and s assignments due to a difference in the cahn
the low energy
lobe gave a negative rotation at 262 nm similar to ( r)-dsp , and ( r)-8-dsi gave a positive rotation
similar to ( s)-dsp .
furthermore , the 8-dsi diastereomers also gave similar
rotations and energies as their analogous dsp diastereomers
at the 238 nm lobe , and 8-dsi and dsp gave similar rotations at the highest energy lobe , but the absolute
energy was different ( i.e. , 202 nm for 8-dsi and
211 nm for dsp ; figure 4 ) .
the similarity in the critical low energy lobe further supports
the absolute configuration assignments that were made from the deamination
studies monitored by hplc .
future computational studies to model these
ecd spectra will help solidify these conclusions , and may address
some of the challenges that occurred during modeling of the ecd spectra
for the dsp diastereomers .
the
ability to deaminate the dsi adducts to dsp allowed the determination of the extinction coefficients
for 5- and 8-dsi relative to dsp , in which these were the values used to determine the relative yields .
peak areas for identically pure 5-dsi samples were measured
before and after incubation in formic acid and the change in peak
area measured was used to determine the relative 240 nm compared to the value established for dsp .
a similar
experiment was conducted for the 8-dsi diastereomers .
from these experiments ,
the 240 nm for the 5-dsi and 8-dsi diastereomers were determined
to be 3800 and 3500 lmolcm , respectively , which are both slightly greater than that determined
for the dsp diastereomers ( 3300 lmolcm ) .
the next step from the nucleoside studies
was to explore the context
dependence of nh3 adduct formation in single- and double - stranded
odns ( ssodn and dsodn ) .
the photooxidant riboflavin was chosen for
the studies because it gave the most diverse distribution of products
in the nucleoside reactions and might provide the most insight into
context effects . for these studies , the ssodns selected for analysis
were the 18-mers odn-1 and odn-2 , and the
dsodn context was studied via the duplex formed from these two single
strands ( odn-12 ) . after oxidation of these odn systems ,
they were digested with a suite of nucleases and phosphatase to the
nucleosides followed by analysis using the previously described hplc
methods .
previously , our laboratory demonstrated that the digestion
method used provides complete degradation of dsp - containing
odns to nucleosides ; it is assumed that
the dsi adducts are equally digested to completion .
the
nuclease digestion conditions were modified to include ammonium salts
for buffer to prevent loss of 5-dsi via deamination to dsp ( scheme 5 ) , controls conducted
with sodium salts for buffers did not detect 5-dsi due
to deamination to dsp during the nuclease digestion ( 18
h at ph 5.4 , scheme 5 ) .
a comparison
between the contexts studied when dg was
allowed to react with nh3 in the presence of the photooxidant
riboflavin is provided in figure 5 .
the first
observation from these data was with respect to dz distributions
that dramatically decreased when proceeding from the nucleoside to
odn contexts ; specifically , the dz distribution in the
dsodn context ( 2% ) was 10-fold less than the ssodn context ( 20% )
and 20-fold less than that observed in the nucleoside context ( 40% ) .
this observation has already been reported in the literature and is
proposed to result from the odn context increasing the lifetime of
the dg that reacts with
nucleophilic h2o to give the c8 product dog while decreasing the lifetime of dg that reacts with o2 to
yield dz ( schemes 2 and 6 ) . with respect to
the yields of the hydantoins , more dsp relative to dgh was observed in the nucleoside context ( 11% vs 4% , respectively )
versus the dsodn context ( 9% vs 21% , respectively ) .
again , this trend
follows those in the literature in which the duplex context favors
the less sterically demanding product dgh .
the nh3 adducts 5-dsi and 8-dsi provided some interesting context - dependent product distributions .
the yield of 5-dsi was greatest for the nucleoside ( 29% )
and decreased by half in all odn contexts studied ( 14% ) . in
contrast
, the yield of 8-dsi was smallest for the nucleoside
( 11% ) and increased by more than 3-fold in the ssodn context ( 39% ) ,
and over half the products observed in the dsodn context were 8-dsi ( 54% ) .
as previously stated , the lifetime of the electrophilic dg is longer in the duplex
context and the superior nucleophilicity of nh3 compared
to h2o greatly increased the yield of 8-dsi in dsodn oxidations ( scheme 6 ) .
furthermore ,
the increase in 8-dsi yield from the dsodn oxidations
further supports this product resulting from nucleophilic addition
of nh3 at c8 leading to 8-dsi , and not the
nh3 aminyl radical adding at c8 to yield the same product .
these results clearly demonstrate a dependence on the reaction context
in formation of dg oxidation products and nh3 adducts .
a look at the distribution of diastereomers resulting from
oxidation of the odn contexts gave nearly equal amounts of both r and s diastereomers .
this observation
parallels our previous work looking at dsp diastereomer
formation in single - stranded and duplex contexts .
coupled together , these observations support a state of
disorder in the duplex at the time of nucleophilic attack at c5 of
oxidized dog or 8-amino - dg ( likely intermediates
leading to the spirocycles ) that leads to nearly equal covalent bond
formation from both the re and si faces of these electrophiles .
relative product distributions measured
when dg was
allowed to react with nh3 in the presence of photoexcited
riboflavin in various contexts .
reactions were conducted in 75 mm
napi buffer ( ph 7.4 ) at 22 c , and 20 mm nh4cl . in
the nucleoside studies , 1
mm dg was studied , and in the
odn contexts 20 m ssodn or 10 m dsodn was studied .
( 1 )
photoactivation of riboflavin ( 200 m ) was achieved with 350
nm light for 3 h in the nucleoside studies and 30 min in the odn studies .
these conditions achieved 70% conversion to product in the
nucleoside reactions and 50% in the odn studies .
it must first be noted
that the bis - ammonium adduct could not be
quantified due to its instability , but if formed , the adduct is most
likely deaminated to either 5-dsi or 8-dsi . keeping this limitation in mind ,
the oxidation of dg by riboflavin initially
yields dg that rapidly
deprotonates to dg in
the nucleoside contexts ( pka
3.9 ) , dominating the product - forming
step of the reaction .
in contrast , dg in the dsodn context retains more cationic character because
the acidic proton is h - bonded with 2-deoxycytosine in a base
pair ; therefore , the product - forming steps of the reaction are determined
by the radical cation intermediate . as shown in scheme 2 , dg could couple
with radicals ( e.g. , o2 ) to
ultimately yield dz , while dg reacts with nucleophiles ( e.g. , h2o and nh3 ) at c8 , ultimately yielding a spirocyclic product
after completion of the four - electron oxidation .
thus , the yield of 8-dsi was anticipated to be greatest in the duplex context ,
as indeed it is .
furthermore , these results support the original hypothesis
by steenken that the duplex context would favor the radical cation
nature of one - electron oxidized dg , which is still the focus of many current research efforts . because of the limitation imposed by the inability to detect the
bis - ammonium adduct , or to understand its decomposition pathway ,
any
comparison between the ammonia adduct distributions would be based
on poorly defined assumptions ; consequently , further discussion of
these results is not provided . for the last set of studies ,
we were inspired by the observation
that the a ring of dsp mimics the h - bonding pattern of
thymidine , while the a ring of 5-dsi mimics the h - bonding
pattern of 2-deoxycytidine ( figure 6 ) . on the basis of this observation , polymerase insertion assays
opposite dsp vs 5-dsi
were conducted to
determine if these h - bonding schemes applied during the selection
of a nucleotide to insert opposite these spirocycles . comparison of h - bonding
patterns between dsp vs t and 5-dsi vs dc .
site - specific synthesis of dsp or 5-dsi was achieved by synthesizing dog into an odn by solid - phase
synthesis and then oxidizing this strand with or without nh4cl present with the one - electron oxidant na2ircl6 to furnish the desired products . upon ion - exchange hplc purification ,
the absolute stereochemical
assignments for the dsp isomers have been established
for the odns , and those for 5-dsi are assigned in this work to be analogous based on the mechanism
of deamination discussed above ( see figure s7 , supporting information , for more details ) .
standing start
polymerase studies were conducted while providing the enzyme only
one type of nucleotide per reaction . on the basis of previous studies ,
insertion of dttp opposite dsp
was not observed ; therefore , to ensure that only one nucleotide
was inserted opposite the lesion , and no extension past the lesions
occurs , the sequence had an a placed 5 to the spirocycle ( figure 7 ) .
klenow fragment exo- was selected as the polymerase ,
and after the extension reactions , polyacrylamide gel electrophoresis
( page ) was conducted to determine the amount of datp , dttp , dgtp ,
or dctp incorporated opposite the dsp or 5-dsi diastereomers .
the polymerase did not select either pyrimidine for
insertion opposite the two lesions ( figure s7 , supporting information ) ; thus , only data for purine insertion
opposite the lesions is provided in figure 7 . with respect to insertion
opposite the dsp diastereomers , ( r)-dsp was observed
to give a slight preference for insertion of datp opposite , and ( s)-dsp gave nearly
equal insertion of datp and dgtp . in comparison to the dsp results
, the 5-dsi isomers showed that ( s)-5-dsi gives similar
amounts of datp and dgtp insertion , while ( r)-5-dsi yields a slight preference
for datp insertion .
unfortunately , these results do not support the
hypothesis that h - bonding in the a ring of the spirocycle is the key
parameter for nucleotide selection , but do show that stereochemistry
may be important for selecting a base pairing partner by this polymerase . sequence
studied for polymerase dntp insertion studies and the
percent dntp insertions opposite the diastereomers of dsp and 5-dsi .
( a ) sequence for the dsodn construct used
in the polymerase insertion assays and ( b ) comparison of the percent
purine dntp insertion opposite the lesions dsp and 5-dsi by the dna polymerase klenow fragment exo- .
the present studies mapped
the pathways and quantified the products
observed when dg was allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose bengal ,
as well as the one - electron oxidant na2ircl6 .
on the basis of the analysis of the products in the nucleoside
context , the major products were oxidant dependent .
the major product
for riboflavin was dz , rose bengal yielded the 5-dsi diastereomers , and na2ircl6 gave
the dsp diastereomers as the major products ( figure 3 ) .
further analyses determined that 5-dsi and 8-dsi decompose via an acid - catalyzed deamination pathway leading to dsp as an end product ( scheme 5 ) .
oxidations
of dg in the nucleoside , ss- and dsodn contexts with
photochemically activated riboflavin demonstrated three major context
effects : ( 1 ) the yield of dz was highest in the nucleoside
studies and decreased dramatically in dsodn contexts ; ( 2 ) when nh3 participated as the nucleophile , 5-dsi was obtained
in the highest yield in nucleoside studies , while the yield of 8-dsi was highest in dsodn contexts ; and ( 3 ) when h2o was the nucleophile in nucleoside reactions , dsp presented
in the highest yield , while in dsodn contexts dgh was
obtained in the highest yield ( figure 4 ) . furthermore ,
a combination of mapping the decomposition pathways of the dsi compounds to dsp , in which the absolute configuration
has been assigned , allowed the determination of the absolute configurations
for the diastereomers of 5- and 8-dsi .
these studies provide fundamental chemical insight
into formation of amine adducts to dg and their stability .
further , these studies insert a cautionary note to researchers who
purify their odns using ammonium salts and then conduct oxidation
reactions after purification .
ammonia is a better nucleophile and
out - competes water for the electrophilic intermediates derived from dg or dog oxidation , resulting in a new mass
of dg + 31 or dog + 15 , respectively .
a similar observation has been highlighted for
oxidations occurring in tris buffer that generate tris adducts to dg .
oxidations were conducted
with 2-deoxyguanosine ( dg ) at 1 mm concentration
in 75 mm napi buffer ( ph 7.4 ) and 22 c .
reactions
with 20 mm nh4cl and without this salt allowed product
profile comparisons .
the oxidants and specific reaction conditions
were achieved as follows : ( 1 ) riboflavin oxidations were initiated
by adding 200 m riboflavin while exposing the samples to 350
nm light for 3 h. ( 2 ) rose bengal oxidations were achieved by adding
100 m rose bengal and exposing these samples to 350 nm for
3 h. the light source for the riboflavin and rose bengal reactions
came from a sun lamp that was placed 7 cm above the reaction
eppendorf tubes .
the tube lids were left open to allow all wavelengths
of light to pass into the reaction samples .
( 3 ) the na2ircl6 oxidations were initiated by a bolus addition of
the oxidizing salt with a final concentration of 10 mm , after a 30
min reaction the samples were quenched with 50 mm edta ( ph 8) .
the
reaction products and their distributions were determined by a dual
hplc method following a previously reported set of protocols ( specific
details can be found in the supporting information file ) . the first rp - hplc run allowed
analysis of dog and 8-amino - dg ( both observed
in < 1% yield ) , while all other products eluted in the void volume
of this run .
the void volume was collected , dried , and reinjected
on a hypercarb hplc column to analyze the diastereomers of dgh , dsp , 5-dsi , and 8-dsi , as
well as the product dz , while monitoring their absorbance
at 240 nm . to determine product distributions the peak areas were
integrated and normalized by each compound s 240 nm ( values provided below ) .
esi - ms , and then each compound
was hplc purified for further structural analysis .
the dgh diastereomers were previously characterized by nmr , dz was also previously characterized by nmr , and the dsp diastereomers have
been characterized by x - ray crystallography .
structural characterization of 5-dsi and 8-dsi by nmr was not conducted because of the instability of these compounds
toward deaminating to dsp , and the lack of nonexchangeable
protons on the ring of either ammonia adducts makes them very challenging
for structural analysis .
furthermore , if deamination of either 5-dsi or 8-dsi to dsp occurs in
the nmr tube during analysis , the peaks observed would be similar
and challenging to interpret ; therefore , the best method for obtaining
structural data on the diastereomers of 5-dsi and 8-dsi is via the esi - ms / ms that was conducted
( figure 2 ) , and provided satisfactory results
to determine their structures .
characterization for each molecule
is as follows , the mixture of the dgh diastereomers ( not
resolvable ) gave tr = 6 min ; lc esi - ms m / z [ m + h ] calcd 274.3 , found 274.1 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c9h15n5o5na 296.0971 , found 296.0979 ; uv vis
240 = 2400 l molcm.(r)-dsp : tr = 11 min ; lc esi - ms m / z [ m + h ] calcd 300.3 , found 300.3 ;
hrms ( esi - tof ) m / z [ m + na ] for c10h13n5o6na calcd 322.0764 , found 322.0761 ; esi - ms / ms m / z [ m + h ] lit .
184 , 156 , 141 , 114 , 113 , 99 , 86 , found 184 ,
156 , 141 , 114 , 113 , 99 , and 86 ; uv vis 240 = 3,300 lmolcm ; cd ( c 1.24 10 m in ddh2o ) nm 259 ( 8.7 ) , 236
( + 35.1 ) , and 211 ( 37.4 ) .
( s)-dsp : tr = 18
min ; lc esi - ms m / z [ m + h ] calcd 300.3 , found 300.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h13n5o6na calcd 322.0764 , found
322.0761 ; esi - ms / ms m / z [ m + h ] lit .
184 , 156 ,
141 , 114 , 113 , 99 , 86 , found 184 , 156 , 141 , 114 , 113 , 99 , and 86 ;
uv vis 240 = 3300 lmol cm ; cd
( c 1.30 10 m in ddh2o ) nm 258 ( + 8.7 ) , 234 ( 32.9 ) , and 212
( + 40.5 ) .
( r)-5dsi : tr = 9 min ; lc esi - ms m / z [ m + h ] calcd
299.3 , found 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0920 ; esi - ms / ms m / z [ m + h ] found values 183 ,
166 , 140 , 123 , 113 , 96 , and 86 ; uv vis 240 = 3800 lmolcm ; cd ( c 1.50 10 m in ddh2o ) nm 258 ( 8.2 ) , 242 ( + 20.8 ) , and 216 ( + 61.8 ) .
( s)-5dsi : tr = 12 min ; lc - esi - ms m / z [ m + h ] calcd 299.3 , found 299.3 ;
hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0916 ; esi - ms / ms m / z [ m + h ] found 183 , 166 ,
140 , 123 , 113 , 96 , and 86 ; uv vis 240 = 3800
lmolcm ; cd
( c 1.45 10 m in ddh2o ) nm 258 ( + 8.2 ) , 242 ( 20.1 ) , and 216
( 62.2 ) .
( r)-8dsi : tr = 7 min ; lc esi - ms m / z [ m + h ] calcd 299.3 , expt 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0924 ; esi - ms / ms m / z [ m + h ] found
183 , 165 , 155 , 138 , 113 , 98 , and 86 ; uv vis 240 = 3500 lmolcm ; cd ( c 1.30 10 m in ddh2o ) nm 261 ( + 20.0 ) , 237 ( 43.2 ) ,
and 203 ( + 18.6 ) .
( s)-8dsi : tr = 14 min ; lc esi - ms m / z [ m + h ] calcd 299.3 , expt 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0926 ; esi - ms / ms m / z [ m + h ] found
183 , 165 , 155 , 138 , 113 , 98 , and 86 ; uv vis 240 = 3500 lmolcm ; cd ( c 1.24 10 m in ddh2o ) nm 262 ( 13.7 ) , 239 ( + 46.0 ) , and 201 ( 33.1 ) .
dz : tr = 27 min ; lc esi - ms m / z [ m + h ] calcd 247.3 , found 247.3 ; hrms ( esi - tof ) m / z [ m + na ] for c8h14n4o5na calcd 269.0862 , found 269.0870 ; esi - ms / ms m / z [ m + h ] lit .
131 and 117 , found 131 and 117 ; uv vis
240 = 1800 lmolcm .
the odns were hplc purified
using an ion - exchange hplc column , and the purification salts ( naoac )
were removed by dialysis prior to their oxidation following previously
reported methods .
the riboflavin oxidations
were conducted similarly to those reported for the nucleoside studies ,
with the following exceptions : the ssodn oxidations were conducted
on 20 m samples while the dsodn oxidations were conducted on
10 m samples . in addition , the reaction times were decreased
for the odn reactions to 30 min .
after the oxidations , the odns were
digested with a suite of nucleases and phosphatases to nucleoside
samples following a previously established protocol , with the exception that all buffers during the digestion
process were comprised of ammonium salts .
next , the digested mixture
was analyzed by the hplc method used in the nucleoside studies .
the polymerase
insertion assays were conducted on duplex odn samples that had site
specific incorporation of dsp or 5-dsi in
the template strand .
the site - specific synthesis was commenced on
odns that had a dog phosphoramidite synthesized at the
desired site of modification within the sequence 5-cgt tax ggc gca act gga aa-3 where x = dog .
the modifications
were synthesized by taking 1 nmol of the dog - containing
odn and placing it in 100 l of reaction buffer ( 75 mm napi , ph 7.4 ) with or without 2 mm nh4cl . the reaction
without nh4cl gave the dsp diastereomers and
the reaction with nh4cl gave the 5-dsi diastereomers .
the individual diastereomers were purified using an ion - exchange hplc
column running naoac as the resolving salt and characterized via digestion
of the odn to nucleosides followed by hplc analysis ( purification
details can be found in the supporting information ) .
the primer template duplex
for the insertion studies was made by annealing 125 nm primer ( 5-tt
tcc agt tgc gcc-3 ) with 156 nm lesion - containing template
( 5-cgt tax ggc gca act
gga aa-3 where x = ( r)-dsp , ( s)-dsp , ( r)-5-dsi , or ( s)-5-dsi to obtain
100 nm duplex in klenow fragment exo- buffer ( 50 mm tris , 50 mm nacl ,
5 mm mgcl2 , 1 mm dtt at ph 8) . to a 25 l reaction
20 l of the annealed duplex was added , 1 l of klenow
fragment exo- ( 0.2 units/l ) , 0.5 l of dntp ( 500 m
stock solution ) , and 8.5 l of klenow buffer to obtain a 100
nm duplex solution with 10 m dntp and 0.2 u of polymerase .
the reaction was incubated at
37 c for 30 min , after which loading dye ( 95% dmf plus 0.025%
bromophenol blue , and 0.025% xylene cylanol ) was added to the samples
and they were heated at 95 c for 20 min to quench the reaction
and denature the dna
the denatured samples
were loaded on a 20% page gel and electrophoresed for 2 h at 45 w.
upon completion of the electrophoresis , the gel was placed in a phosphor
screen overnight and imaged by storage phosphor autoradiography . | upon oxidation of the heterocyclic
ring in 2-deoxyguanosine
( dg ) , the initial electrophilic intermediate displays
a wide range of reactivities with nucleophiles leading to many downstream
products . in the present study ,
the product profiles were mapped when
aqueous solutions of dg were allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose
bengal as well as the diffusible one - electron oxidant na2ircl6 .
product characterization identified the 2-deoxyribonucleosides
of spiroiminodihydantoin , 5-guanidinohydantoin , and oxazolone resulting
from h2o as the nucleophile .
when nh3 was the
nucleophile , a set of constitutional isomers that are diastereotopic
were also observed , giving characteristic masses of dg + 31 .
esi+-ms / ms of these nh3 adducts identified
them to be spirocycles with substitution of either the c5 or c8 carbonyl
with an amine .
the nh3 adducts exhibit acid - catalyzed hydrolysis
to spiroiminodihydantoin .
quantification of the nh3 and
h2o adducts resulting from oxidation of dg in the nucleoside , single - stranded , and duplex oligodeoxynucleotide
contexts were monitored allowing mechanisms for product formation
to be proposed .
these data also provide a cautionary note to those
who purify their oligonucleotide samples with ammonium salts before
oxidation because this will lead to unwanted side reactions in which
ammonia participates in product formation . | Introduction
Results and Discussion
Conclusion
Experimental
Section |
PMC4483387 | ankle dorsiflexion passive range of motion ( df prom ) measurements are performed in the
field of physical therapy to estimate ankle motion during functional activities1 and to prevent lower extremity injuries2 .
although in the clinical setting , ankle df
prom is frequently measured under non - weight - bearing ( non - wb ) conditions1 , 3 , 4 , many researchers have stated that the wb
position is more appropriate for estimating the amount of ankle df motion during functional
activities5 , 6 . therefore , wb ankle df prom should be measured during interventions
focused on increasing ankle df prom .
limited ankle df prom with knee extended may result from gastrocnemius tightness and
insufficient posterior talar glide7 .
thus ,
gastrocnemius stretching and talocrural joint mobilization have been performed as
intervention strategies to increase ankle df prom3 ,
8 , 9 .
previous studies have reported a significant increase in ankle df
prom after these interventions3 , 8 , 9 ; however , to our
knowledge , no study has demonstrated the combined effect of both interventions on wb ankle
df prom .
therefore , the aim of the present study was to examine the influence of
gastrocnemius stretching combined with joint mobilization on wb ankle df prom .
in total , 11 male subjects with bilateral limited non - wb ankle df prom with knee extended
( mean age , 22.82 3.09 years ; mean height , 175.91 3.39 cm ; mean weight , 69.55 3.78 kg ;
mean non - wb ankle df prom , 4.17 2.48 ) participated in this study .
inclusion criteria were
1 ) ankle df prom with knee extension < 10 ; 2 ) ankle df prom with knee flexion > 10 ;
and 3 ) > 5 difference in ankle df prom between knee extension and knee flexion
conditions on bilateral sides in non - wb positions3 .
subjects with a history of surgery on the lower extremity ,
fracture , or neurological diseases were excluded from this study .
all participants signed an
informed consent form approved by the institutional research review committee of inje
university prior to participation in this study .
the study protocol of this study complies
with the ethical standard of the declaration of helsinki .
wb ankle df prom with knee extended was measured following the procedures suggested by
munteanu et al10 .
subjects stood in front
of a wall and placed the leg being tested behind the contralateral leg in a lunge posture .
subjects were asked to place both hands on the wall and then lean forward without heel - off
and knee flexion until maximum stretch was felt in the gastrocnemius on the tested leg .
the
force applied to the tested leg was maintained at 60 5% of the subject s weight using
scales11 .
an examiner determined the
maximum tibial inclination using an inclinometer to measure the wb ankle df prom with knee
extended .
measurements of wb ankle df prom were repeated 3 times for each ankle under pre-
and post - intervention conditions .
the mean value of 3 trials was used for data analysis . for gastrocnemius stretching combined with joint mobilization , subjects leaned forward
against the wall in the same lunge posture as that during measurement of wb ankle df prom
with knee extended until the maximum gastrocnemius stretch was felt .
subjects held the
end - range posture while an examiner provided the talus of the tested leg with sustained
anterior - to - posterior gliding force .
an intervention trial was performed for 30 s , and 10
trials were repeated with 30-s rest periods for each ankle . the difference in wb ankle df prom with knee extended between pre- and post - intervention
conditions was analyzed using a paired t - test .
wb ankle df prom with knee extended was significantly increased in post - intervention
compared with pre - intervention conditions ( 42.60 5.49 versus 38.24 4.69 , p <
0.001 ) .
our findings demonstrate that gastrocnemius stretching combined with joint mobilization
significantly increases wb ankle df prom with knee extended . stretching exercises
increase
tolerance , resulting in increased rom12 .
additionally , increased displacement of the myotendinous junction ( mtj ) after gastrocnemius
stretching for 5 min was found in a previous study13 .
therefore , the change in tolerance and/or increase in mtj
displacement might have influenced our findings .
the addition of talocrural joint
mobilization to gastrocnemius stretching is another possible explanation for our findings .
previous research by dinh et al.3 showed a
4.25 increase in wb ankle df prom with knee extended after gastrocnemius stretching alone
for 3 weeks . although gastrocnemius stretching combined with joint mobilization
was applied
for 5 min in the present study , the amount of increase in wb ankle df prom after
intervention ( i.e. , 4.36 ) was similar to that found previously . considering this outcome ,
despite the relatively short period of intervention in the present study , it may be inferred
that the addition of talocrural joint mobilization might maximize the effects of general
gastrocnemius stretching .
thus , we
conclude that gastrocnemius stretching combined with joint mobilization might decrease
gastrocnemius tightness and increase posterior talar gliding movement , which effectively
increases wb ankle df prom with the knee extended .
first , although non - wb ankle df prom was used as
an inclusion criterion , changes in non - wb ankle df prom after intervention were not
measured .
however , we believe that wb ankle df prom is clinically more important because
most functional activities are performed under the wb condition .
second , our study included
only male subjects , and the results can not be generalized to women . | [ purpose ] the purpose of this study was to investigate the effect of gastrocnemius
stretching combined with talocrural joint mobilization on weight - bearing ankle
dorsiflexion passive range of motion .
[ subjects ] eleven male subjects with bilateral
limited ankle dorsiflexion passive range of motion with knee extended participated in this
study .
[ methods ] all subjects received talocrural joint mobilization while performing
gastrocnemius stretching .
ankle dorsiflexion passive range of motion was measured using an
inclinometer under weight - bearing conditions before and immediately after intervention .
a
paired t - test was used to analyze the difference between weight - bearing ankle dorsiflexion
passive range of motion pre- and post - intervention .
[ results ] a significant increase in
weight - bearing ankle dorsiflexion passive range of motion was found post - intervention
compared with pre - intervention .
[ conclusion ] these findings demonstrate that gastrocnemius
stretching combined with joint mobilization is effective for increasing weight - bearing
ankle dorsiflexion passive range of motion . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
PMC2671006 | in the past 15 years , green fluorescent protein ( gfp ) has changed from a nearly unknown protein to a commonly used molecular imaging tool in biology , chemistry , genetics , and medicine . in 2006 , more than 10 000 papers
gfps and gfp - like proteins ( i.e. , chromoproteins and fluorescent proteins ) are particularly useful due to their stability and the fact that the chromophore ( see figure 1 ) is formed in an autocatalytic cyclization of the 65syg67 sequence that does not require a cofactor .
this means that unlike most other bioluminescent reporters , gfp fluoresces in the absence of any other proteins , substrates , or cofactors .
furthermore , it appears that fusion of gfp to a protein does not alter the function or location of the protein . by changing residue 66 and/or the amino acid residues around the chromophore
(n1c1c2c3 ) and (c1c2c3c4 ) dihedral angles of the gfp chromophore . in the protein ,
r1 is gly67 and r2 is ser65 , and in hbdi , an often - used model compound , r1 = r2 = ch3 . in one - bond flips ( -obf ) the dihedral rotation occurs around the torsional angle , in a -obf it is around the dihedral angle , and in a positively correlated hula - twist ( + ht ) the and dihedral angles concertedly rotate in the same direction ( as shown above ) , while in a negatively correlated hula twist ( ht ) they concertedly rotate in opposite directions . a plot of the and dihedrals for a perfectly correlated negative ht will have a slope of 1 .
if the chromophore cavity is complementary with a planar chromophore , then the and best fit line will pass through the origin and all the and angles will be centered around the origin .
a nonzero intercept along the or axis ( see , for example , figure 4 ) or and dihedrals centered in quadrant ii ( > 0 ;
< 0 ) or quadrant iv ( < 0 ; > 0 ; see , for example , figure 8) are indications of a cavity that is not complementary with a planar chromophore . the fluorescent emission of the chromophore within gfp occurs with high efficiency ( quantum yield fl = 0.8 ) and a respectable fluorescence lifetime ( 3 ns).(19 ) when the protein is denatured the fluorescence yield decreases by at least 3 orders of magnitude.(20 ) model compounds of the chromophore do not fluoresce in solution ( quantum yield fl < 10 ) , unless the rotation of the arylalkene bond is restrained.(21 ) fluorescence can , however , be obtained by lowering the temperature to 77 k ; this freezes the solution .
it has been suggested that twisting between the phenolate and imadazolidinone groups of the chromophore is the mechanism for the ultrafast fluorescence quenching internal conversion process .
neutral form ( a ) of the chromophore , with the phenolic oxygen protonated , can convert to the anionic species ( b ) by going through an intermediate state ( i ) .
the change from forms a to i is solely a protonation change , while the change from i to b is a conformational change with most changes occurring at thr203 .
upon excitation of the a state , an excited - state proton transfer ( espt ) occurs in which the proton is transferred from the chromophore to glu222 in a time scale of the order of picoseconds . following radiative relaxation from the excited - state intermediate ( i * ) ,
the systems returns to the ground state a through the ground - state intermediates i1 and i2.(29 ) excitation of the anionic b state results in direct emission from the excited state ( b * ) at 482 nm . recently
, a nonfluorescent dark state , state c , has been observed that is distinct from states a and b and absorbs at higher energies.(30 ) the c state , perhaps the neutral trans form of the chromophore , may be populated by nonradiative decay from a * and it may be depopulated by excitation to the excited c * state with transcis isomerization to repopulate state a. fluorescent blinking has been ascribed to nonadiabatic crossing and conversions between the neutral and anionic states,(31 ) or a possible dark ( z ) zwitterionic state . while the ground - state minimum of the gfp chromophore is close to planar , this is not necessarily so for the excited state ; in fact , in some cases the excited state has an energy minimum with a twisted chromophore in which both rings are 90 to each other.(32 ) according to quantum mechanical calculations , the ground and excited states for the one - bond flip ( obf ) and hula twist ( ht ) in the neutral form ( a ) and the obf in the zwitterionic form come very close to each other .
it has been proposed that in the absence of the protein matrix , which surrounds the chromophore and prevents twisting , this process can lead to fluorescence quenching internal crossing;(32 ) see figure 3 .
recent calculations on the gfp chromophore model compound hbdi suggest that the anionic form of hbdi may also undergo a -obf that leads to a favored radiationless decay channel , which is particularly efficient in solvent.(34 ) ( a ) model compounds of the gfp chromophore in the ground state ( s0 ) can be excited to the first singlet state ( s1 ) in which a ht or obf can freely occur . upon reaching the perpendicularly twisted conformation , fluorescence quenching nac ( nonadiabatic crossing ) occurs .
( b ) in the ground state ( s0 ) , the residues surrounding the gfp chromophore exert a twisting force on the chromophore ( ) . upon excitation , the conjugation across the ethylenic bridge of the chromophore is reduced and it will twist ; however , the protein matrix prevents the chromophore from reaching the perpendicularly twisted conformation ( ) and fluorescence quenching internal crossing is prevented .
recently , the results from a number of interesting experiments that provide evidence for chromophore twisting and/or fluorescence quenching internal crossing ( ic ) have been published .
for example , a molecular dynamics simulation of the chromophore of cyan fluorescent protein found that the average dihedral angle was about 0 ; however , when the surrounding protein was considered in the simulation the average shifted by approximately 5 , showing that the protein matrix of cfp twists the chromophore . on the basis of their calculations , the authors concluded that the driving force for this twist comes from the strong short - range repulsion by four residues ( ile167 , val150 , phe165 , and thr203 ) surrounding the average position of the chromophore.(46 ) photoswitching fluorescent proteins can be switched back and forth between the naturally occurring green state and a dark state by 405 nm irradiation ( e.g. , dronpa , mtfp0.7 , kfp1 ) .
a cistrans isomerization of the chromophore has been proposed as the structural basis for the photoswitching observed in dronpa.(47 ) this is supported by the fact that mutating either val157 or met159 with smaller residues accelerates photoswitching , presumably by decreasing steric hindrance to cistrans isomerization.(48 )
the m159 t and v157 g mutations also decrease the quantum yield of dronpa from 0.85 to quantum yields of 0.23 and 0.77 , respectively.(48 ) recently , it has been suggested that adoption of a trans configuration can not solely be responsible for the nonfluorescent form.(49 ) on the basis of nmr analyses , miyawaki et al .
propose that the fluorescence of the protein is regulated by the degree of flexibility of the chromophore but is not necessarily accompanied by cistrans isomerization.(49 ) interestingly , gfp is not unique in photoactive yellow protein ( pyp ) the protein matrix also prevents the chromophore from adopting a completely planar structure . in pyp
the asymmetric proteinchromophore interaction probably serves as the initial accelerant for the light induced photocycle,(50 ) which ultimately leads to a cistrans isomerization.(51 ) the chromophore in wild - type gfp is planar due its extended system ( see figure 1 ) ; however , the energy barrier to deformation is low and the protein matrix in wild - type gfp exerts some strain away from planarity .
when the chromophore is computationally permitted to freely rotate , it will adopt a conformation that complements the protein matrix .
recently , we have used computational methods to show that wild - type gfp is not an anomaly and that all gfp and gfp - like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore.(54 ) in most cases the freely rotating chromophore undergoes rotations of at least 20. in some cases these rotations are accompanied by an equal but opposite rotation of the dihedral angle ( a negatively correlated ht ) .
none of the proteins examined have a cavity that causes a rotation solely around the dihedral angle.(54 ) these calculations were done by minimizing , with freely rotating and dihedral angles , the crystal structure of 38 gfp analogues and mutants found in the pdb .
they found the energy minimum conformation of a freely rotating chromophore in the protein matrix of the gfp mutant or gfp - like protein examined ; however , they did not provide any information about the range of low - energy conformations available to a freely rotating chromophore . to get this information
, we ran molecular dynamics simulations of some of the interesting gfp mutants in the protein databank.(56 ) by running molecular dynamics simulations , with freely rotating and dihedrals , we have been able to determine the range of conformations available to chromophores with complete rotational freedom .
the coordinates of three crystal structures ( 1gfl,(57 ) 1myw,(58 ) 2emd(59 ) ) were obtained from the protein data bank ( pdb),(56 ) and hydrogen atoms were added to protein and solvent atoms as required . the opls_2005 force field of macromodel v9.0016(60 ) was used .
starting structures for mutants for which no crystal structure has been determined were calculated by graphically mutating a known structure and undertaking a conformational search .
the flexible dihedral angles of all the side chains of residues that are within 8.00 of the chromophore were randomly rotated by between 0 and 180 , and all solvent molecules in that sphere were randomly rotated and translated by between 0 and 1.00 in each monte carlo ( mc ) step.(63 ) 15 000 mc steps were taken in each search .
structures within 50 kj / mol of the lowest energy minimum were kept and a usage - directed method(62 ) was used to select structures for subsequent mc steps .
structures found in the conformational search were considered unique if the least - squared superimposition of equivalent non - hydrogen atoms found one or more pairs separated by 0.25 or more . the lowest energy structure obtained in the monte carlo torsional and molecular position variation search
the final structures obtained from the mc search ( monte carlo torsional and molecular position variation and large - scale low mode ) or fully minimized pdb structures were used to initiate md simulations with freely rotating and dihedral angles ( v1 = v2 = v3 = 0.000 ) .
the predynamics simulation was set for 100 ps and the full md simulation for 5000 ps .
the cambridge structure database ( csd ) v5.29 was released in january 2008 ; it comprises 436 384 small molecule crystal structures .
the area of the convex hull of the and graphs was calculated as was the smallest convex set containing the region .
molecular mechanics and dynamics calculations have been used to examine the steric environment of the chromophore in gfp in its ground state .
they are techniques that are based on classical physics and were designed to model structural and not electronic properties ; therefore , molecular mechanics and dynamics simulations of gfp can not examine the excited state of the chromophore .
quantum calculations are an excellent technique to determine the energy profiles of the ground and excited states of the chromophore ; however , they are cpu - intensive and it would be cost prohibitive to do conformational searches on the excited state of a series of gfp and gfp - like proteins .
therefore , in an attempt to supplement the quantum calculations , we have examined the conformational space available to the chromophore within gfp using a freely rotating chromophore that is an approximation ( based on qm calculations ) of the conformational space available to the chromophore in its excited state . as shown in figure 2 ,
gfp adopts two states , the neutral state ( a ) of the chromophore with the phenolic oxygen protonated , and the anionic species ( b )
the change from forms a to i is solely a protonation change , while the change from i to b is a conformational change with most changes occurring at thr203.(67 ) in order to see whether the gfp chromophore has different dihedral freedom in forms a , b , and i , we conducted molecular dynamical simulations with a freely rotating chromophore , as described in the .
not surprisingly , table 1 and figure 4 show that forms a , b , and i of gfp have similar dihedral freedom and that all undergo similar negatively correlated ( same slopes of best fit lines in figure 4 ) hula twists .
plot of the vs dihedral angles ( see figure 1 for nomenclature ) for the 2000 gfp - a ( pink ) , gfp - b ( green ) , and gfp - i ( light - blue ) structures obtained in the freely rotating molecular dynamics simulation . however , there are subtle differences ; forms a and i are closer to the planar ground - state conformation , while the b form adopts conformations further from planarity when given rotational freedom around the and dihedrals ( intercept further from = 0 ) ; and the anionic b form has the most freedom ( largest area , and and range ) , while the neutral a form has the least freedom .
the a form has robust hydrogen bonds between both arg96 and gln94 and the imidazolidinone carbonyl group that remain intact throughout the simulations . in the intermediate ( i )
form these hydrogen bonds are supplemented by an additional hydrogen bond between the phenolic oxygen of the chromophore and his148 ( see table 2 ) .
the main difference between the anionic b form and the intermediate form is that thr203 has to rotate in order to hydrogen bond to the phenolic oxygen in form i , otherwise they have the same hydrogen - bonding interactions throughout the simulation .
the y66h mutant of gfp exhibits blue fluorescence and has therefore been termed blue fluorescent protein .
the crystal structure of bfp ( not to be confused with blue fluorescent protein from aequorin bfp - aq(68 ) ) has been solved at ph 4.5(27 ) and ph 8.5;(59 ) the overall fold of the protein is identical to wild - type gfp and consists of a chromophore surrounded by an 11-stranded -barrel .
while gfp has absorption maxima at 395 and 475 nm and emits at 508 nm , bfp absorbs at 382 nm and emits at 448 nm.(9 ) unfolding of bfp results in an absorption red shift of 15 nm,(27 ) and quantum mechanical calculations suggest that the 15 nm shift might be due to the protein - induced nonplanarity of the chromophore.(69 ) blue fluorescent protein ( bfp ) has a much lower fluorescence quantum yield than gfp ( fl = 0.20 vs 0.80 ) .
it has been suggested that this is due to the fact that his66 ( bfp - chromophore ) forms fewer hydrogen bonds with the surrounding protein than tyr66 ( gfp - chromophore ) does , and that the smaller imadazole ring ( his66 ) in bfp may have more conformational freedom than the larger phenol ( tyr66 ) , which leads to more intersystem crossing.(27 ) in a very elegant series of experiments , boxer et al .
have shown that the fluorescence quantum yield of blue fluorescent protein increases from fl = 0.20 to 0.35 when the pressure is increased from atmospheric pressure to 570 mpa.(70 ) analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes .
temperature - dependent fluorescence measurements reveal two barriers ( 19 and 3 kj / mol , respectively ) for the transition into nonfluorescing states .
these steps are probably linked with dissociation of the hydrogen bond between the chromophore and his148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state , respectively.(70 ) in order to establish the consequences of the y66h mutation on the flexibility of the chromophore , molecular dynamics simulations of the ph 8.5 bfp crystal structure ( pdb code 2emd with neutral imidazole rings for his66 and his148 ) with a freely rotating chromophore were run .
[ we thank one of the referees for suggesting the ph 8.5 ( 2emd ) over the ph 4.5 ( 1bfp ) structure .
] figure 5 and table 1 show that the chromophore in bfp has significantly more rotational freedom than that available to the chromophore in gfp , and that the bfp cavity is less complementary to a planar chromophore than the gfp cavity is ; that is , the bfp sampled structures are further from a planar chromophore ( = = 0 ) and the intercept for the best fit line through the bfp structures is further from the origin than that of gfp .
plot of the vs dihedral angles for the 2000 bfp ( blue ) and gfp - a ( green ) structures obtained in the freely rotating molecular dynamics simulation .
analysis of the 2000 bfp structures revealed a hydrogen bond between the nh of the his66 imidazole ring and glu222 , which is retained through - out the entire simulation ( see figure 7 ) .
the smaller imadazole ring of the bfp chromophore results in more dihedral freedom for the chromophore in bfp than phenol in gfp ; however , there seems to be no large difference in the number and stability of hydrogen bonds formed by the chromophore in the two fps .
recently , two new blue fluorescent proteins ( azurite and a5 ) with enhanced brightness and photostability were created .
the methodology applied to find the brighter bfp mutants was based on the concept that replacing the residues surrounding the chromophore with bulkier amino acids would constrain the chromophore s motion and thereby increase the proteins brightness .
the crystal structure of a5 has yet to be solved . in order to find a starting conformation for the md simulation , the 2emd crystal structure
was graphically mutated to a5 and a thorough conformational search was undertaken ( see ) .
given the high structural similarity between all the solid state structures of gfp mutants in the protein databank , a thorough conformational search should find the lowest energy conformations of a5 by making the relevant mutations to the 2emd structure .
figure 6 and table 1 show that the conformational space available to the chromophore in bfp is larger than that in a5 and that the bfp conformations tend to be further from planarity ; i.e. , for a5 median = 2.92 , median = 2.22 and intercept of best - fit line is 4.62 vs for bfp median = 27.59 , median = 10.18 and intercept of best - fit line 14.27 .
similar results were found for md simulations carried out from all of the five lowest energy conformational families sampled in the conformational search of a5 and in a md simulation initiated from an a5 structure obtained by taking the 2emd structure , graphically mutating it to a5 , and thoroughly minimizing the structure without undertaking a conformational search .
plot of the vs dihedral angles for the 2000 bfp ( dark blue ) and a5 ( pink ) structures obtained in the freely rotating molecular dynamics simulation .
the n of the his66 imidazole ring hydrogen bonds with water305 ( 2emd numbering ) in all the sampled structures .
this water connects his66 to the surface of the protein via a robust hydrogen - bonding network to his148 and is also hydrogen bonded to glu222 . in half the structures sampled , the n of the his66 imidazole ring hydrogen bonds to water302 which is in an extensive hydrogen - bonding network with val68 , arg224 , and gln69 .
in contrast to 2emd there are no hydrogen - bonding interactions with the n hydrogen ; see figure 7 .
hydrogen - bonding interactions ( --- ) for 2emd ( top ) and a5 ( bottom ) chromophore .
these results seem to indicate that the enhanced brightness of a5 is at least in part due to the fact that its chromophore movement is restricted relative to bfp .
this restriction can be due to both steric factors and the increased hydrogen - bonding networks in a5 . in the first rationally designed mutant based on the crystal structure of gfp - s65 t ,
it was decided to mutate t203 into a tyrosine so that it could stack with the phenolic group in the chromophore .
( 73 ) the resultant mutant , yfp , is red - shifted by 16 nm relative to gfp - s65 t and does indeed have a stacking interaction between the chromophore and tyr203.(74 ) on the basis of the crystal structure of yfp , remington et al .
proposed that the red shift was due to the additional polarizability of the -stacked tyr203 , the hydrogen - bond pattern around the chromophore , or an out - of - plane distortion of the chromophore ( in analogy with out - of - plane distortions in porphyrin systems).(74 ) comparison with the t203v mutant revealed that the t203y substitution leads to a significant i - form population and only about 10 nm of the shift can be ascribed to the interaction.(75 ) a new varient of yfp , venus , with improved brightness and maturation properties as well as a reduced environmental sensitivity was developed and crystallized.(58 ) since some researchers have argued that the presence of tyr203 leads to a decrease in chromophore flexibility ( van der waals volume for tyr = 141 vs 93 for thr ) , and others argue that this is not necessarily so,(32 ) we have examined the conformational space available to a freely rotating chromophore in yfp ( pdb code 1myw ) with the phenol of tyr66 in both the protonated and unprotonated forms . both table 1 and figure 8
show that the chromophore ( in the phenolic form ) in gfp and yfp has differing rotational freedom . in yfp
the chromophore has approximately 1.2 times more dihedral freedom than in gfp and although the dihedrals undergo negatively correlated hula - twisting in both proteins , in yfp the average conformation sampled is further from planarity than that found in gfp ; i.e. , the majority of the sampled conformations are located in quadrant iv .
plot of the vs dihedral angles ( see figure 1 for nomenclature ) for the 2000 structures obtained in the freely rotating molecular dynamics simulation of gfp ( dark blue ) and yfp ( pink ) in their phenolic forms .
the majority of the yfp conformations sampled are nonplanar ( i.e. , in quadrant iv < 0 and > 0 ) . during the entire simulation the hydrogen bonds between tyr66 and ser205 , arg96 and the imidazolone carbonyl , as well as between tyr203 and water354 remain stable , while those between tyr203 and gln69 were less stable .
we also examined the distance between the centroid of tyr66 ( the choromophore ) and tyr203 .
the distance is fairly short and does not change much during the simulation ( minimum = 3.43 , maximum = 4.18 , average = 3.75 , sd = 0.103 ) while the angle between the planes of the phenols varies between 0.0 and 22.5 ( average = 6.9 , sd = 4.11 ) .
the short distance between the phenol rings is indicative of stacking , which may be responsible for the red shift observed in yfp ; however , the variability in the angle between the planes of the phenols shows that tyr203 does not significantly restrict the rotational freedom of the excited chromophore .
the cambridge structural database ( csd)(77 ) v5.29 comprises 436 384 small molecules crystal structures .
it has 5551 structures with two phenol rings in separate molecules that have centroids within 5.00 of each other .
the closest ones are 3.16 from each other , and only 832 structures are within 3.75 ; i.e. , the average distance between the phenol centroids in the freely rotating yfp .
a plot of the distance vs the angle between the phenol planes in our freely rotating simulation and the csd structures shows that the protein matrix of yfp restricts the distance between tyr66 and tyr203 from being larger than 4.18 , but that the angles between the two phenols have as much freedom in yfp as in the small molecule structures with intermolecular distances of less than 4.18 ( see figure 9 ) .
it does not seem as if the increased fluorescence lifetime of yfp is due to a decrease in the dihedral freedom of the chromophore .
it is more likely due to electronic effects such as those proposed by jung et al.(78 ) they suggested that the anionic form of the chromophore can be described by two mesomeric forms , the benzoidal and the quinoidal structure .
the benzoidal form with the majority of its negative charge on the phenol is stabilized by hydrogen bonding to thr203 , which is not possible in yfp and therefore the t203y mutation favors the quinoidal form , which may have a longer fluorescence lifetime.(78 ) distance vs angle between the phenol planes in yfp ( ) and all structures in the csd that have two unconnected phenols separated by less than 5.00 ( + ) .
some have suggested that the crystal structures of strongly fluorescent gfp and gfp - like proteins have their chromophores in a cis configuration , while those of the nonfluorescent proteins are in a trans configuration .
however , the trans - planar form of eqfp611 is an important exception : it is fluorescent.(82 ) remington et al .
have suggested that coplanarity might be more important than isomer configuration in determining the efficiency of fluorescence .
most current theories ( see ) suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield .
our calculations show that this is the case for the systems examined . for bfp , a5 , yfp , and gfp
, there is an inverse correlation between the dihedral freedom of the chromophore and the quantum yield ; see table 1 . in all simulations ,
the protein matrix is not complementary with a planar chromophore , and in all cases the freely rotating chromophore undergoes a negatively correlated hula twist ( also known as a bottom hula twist mechanism ) . | green fluorescent protein ( gfp ) and gfp - like fluorescent proteins owe their photophysical properties to an autocatalytically formed intrinsic chromophore . according to quantum mechanical calculations ,
the excited state of chromophore model systems has significant dihedral freedom , which may lead to fluorescence quenching intersystem crossing .
molecular dynamics simulations with freely rotating chromophoric dihedrals were performed on green , yellow , and blue fluorescent proteins in order to model the dihedral freedom available to the chromophore in the excited state .
most current theories suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield . according to our calculations , the dihedral freedom of the systems studied ( bfp > a5 > yfp > gfp ) increases in the inverse order to the quantum yield . in all simulations ,
the chromophore undergoes a negatively correlated hula twist ( also known as a bottom hula twist mechanism ) . | Introduction
Experimental Section
Results and Discussion
Conclusion |
PMC3279530 | an expanded research design and methods section and supplementary figures can be found in an online data supplement available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1293/dc1 .
details on glucose and insulin tolerance testing , plasma insulin level determination , lipid metabolite measurement , metabolomics , exercise capacity studies , whole - body in vivo metabolic assessment , and immunoblot analysis are provided in the online data supplement .
all animals received care according to the canadian council on animal care and the university of alberta health sciences animal welfare committee .
twelve - week - old c57bl/6 mice were placed on a standard chow / low - fat diet ( 4% kcal from lard ) or high - fat diet ( 60% kcal from lard , research diets ; d12492 ) for a 12-week period . at the end of week 12
, animals were injected intraperitoneally every other day with the spt1 inhibitors , myriocin ( 0.5 mg / kg ) suspended in 1x pbs , l - cycloserine ( 25 mg / kg ) suspended in 1x pbs , or vehicle control for a 4-week period . at the end of the 4-week treatment protocol
, animals were killed via intraperitoneal injection of sodium pentobarbital ( 12 mg ) in the fed state in the middle of the dark cycle .
tissues were excised and immediately frozen in liquid n2 . in another study , 6-week - old db / db mice and their heterozygous controls ( db/+ ) ( jackson laboratories )
as expected , mice fed a high - fat diet for 12 weeks became obese as indicated by a significant increase in weight gain , ( supplementary fig .
d ) . diet - induced insulin - resistant and lean mice were placed in a comprehensive lab animal - monitoring system ( clams ) for whole - body metabolic assessment , which demonstrated a high - fat diet induced shift in fuel preference toward fatty acids as an oxidative energy source , indicated by the large drop in the respiratory exchange ratio ( rer ) ( supplementary fig .
further support for an increase in fatty acid oxidation in obese mice is seen with the increase in gastrocnemius -hydroxyacyl - coa dehydrogenase ( had ) activity ( table 1 ) .
contrary to previous findings ( 16,17 ) , we also report here that obesity induced by chronic high - fat feeding impairs whole - body oxygen consumption rates ( supplementary fig . 2c and d ) .
-hydroxyacyl - coa dehydrogenase ( had ) activity in gastrocnemius muscle of lean and obese mice treated with vehicle control or myriocin values reported are mol / g wet weight / min of n = 5 mice .
differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart .
after 12 weeks of low- or high - fat diet , mice were treated with either myriocin ( 0.5 mg / kg every other day ) or vehicle control .
after 2 weeks of treatment , we demonstrated that inhibition of spt1 with myriocin reverses diet - induced insulin resistance , as determined by glucose tolerance and insulin tolerance testing ( fig .
if these protective effects took place at the skeletal muscle level , a group of animals were killed at 30 min after insulin injection during the insulin tolerance test , and muscles were excised and harvested for immunoblot analysis of the insulin - signaling pathway .
we demonstrate that insulin stimulation of both akt and glycogen synthase kinase 3 ( gsk3 ) phosphorylation were significantly improved in the gastrocnemius muscle of obese mice treated with myriocin ( fig .
phosphorylation of 5amp activated protein kinase ( ampk ) , another key signaling molecule regulating glucose metabolism , did not differ in gastrocnemius muscle of control and myriocin - treated obese mice ( data not shown ) .
induced insulin resistance and improves insulin signaling . a : glucose tolerance test in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin .
c : insulin tolerance test in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin .
e : insulin - stimulated akt phosphorylation at serine 473 , and ( f ) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of obese insulin - resistant mice treated with either vehicle control or myriocin .
values represent mean se ( n = 812 for a d ; n = 4 for e and f ) .
differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from all other groups .
myriocin treatment was without effect on food intake , body weight , and plasma insulin levels , but did reduce both postprandial and fasted plasma glucose levels in obese , insulin - resistant mice ( table 2 ) . although fasting plasma insulin levels did not differ between diet - induced obesity ( dio ) mice treated with vehicle - control or myriocin , more sophisticated studies monitoring the changes in plasma insulin in response to a meal tolerance test in the obese jcr : la cp rat illustrate a significant improvement in plasma insulin control after treatment with the spt1 inhibitor , l - cycloserine ( supplementary fig .
3 ) . interestingly , indirect calorimetry revealed that the improved insulin sensitivity in dio mice treated with myriocin was not associated with a decrease in fatty acid oxidation and an increase in carbohydrate oxidation , as similar rer values were observed between the dio control and myriocin - treated animals ( fig .
2 ) . effect of myriocin on body and tissue weight , and plasma glucose and insulin levels in lean and obese mice values reported are from n = 511 mice .
differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart ; significantly different from high - fat control .
twenty - four - hour ( a ) , dark cycle ( b ) , and light cycle respiratory exchange ratio ( c ) in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin .
differences were determined using a two - way anova followed by a bonferroni post hoc analysis .
* p < 0.05 , significantly different from the low - fat diet counterpart . in a parallel series of experiments , after 12 weeks of high - fat feeding , mice were treated with either the spt1 inhibitor , l - cycloserine ( 25 mg / kg every other day ) or vehicle control . although not as dramatic as the results observed with myriocin , at 2 weeks after treatment , we report improvements in glucose and insulin tolerance in mice treated with l - cycloserine ( supplementary fig .
obese , insulin - resistant mice were run on an exercise treadmill to determine exercise capacity . as expected , obese mice showed a dramatic reduction in both their treadmill time and distance when compared with their lean counterparts ( fig .
interestingly , treatment of obese mice for 2 weeks with myriocin reversed this reduction in exercise capacity ( fig . 3a and b ) .
this improvement in exercise capacity observed in obese mice treated with myriocin can be explained by enhanced whole - body oxygen consumption rates compared with their control counterparts ( fig .
c ) . in addition , we observed greater citrate synthase activity in gastrocnemius muscle of obese mice treated with myriocin ( fig .
protein expression of peroxisome proliferator - activated receptor- coactivator-1 ( pgc1 ) , a transcriptional coactivator that plays a key role in regulating a number of genes involved in energy metabolism ( 18 ) , showed a trend toward a reduction in control - treated dio mice ( p = 0.077 ) that was not apparent in myriocin - treated dio mice ( fig .
furthermore , we also demonstrate that pretreatment with myriocin increases citrate synthase activity in c2c12 myotubes exposed to 1.0 mmol / l palmitate for 16 h ( fig .
these observations illustrate improvements in mitochondrial function , possibly explaining why exercise capacity and whole - body oxygen consumption rates were enhanced in this group .
time ( a ) and distance ( b ) during an exercise capacity challenge on a running treadmill .
differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart .
myriocin treatment reverses the impairment in whole - body oxygen consumption rates caused by dio .
c : twenty - four hour ( a ) , dark cycle ( b ) , and light cycle ( c ) whole - body oxygen consumption assessment in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin .
d : gastrocnemius muscle citrate synthase activity in vehicle control and myriocin - treated dio mice .
e : pgc1 expression in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin .
f : citrate synthase activity in vehicle control and myriocin - pretreated c2c12 skeletal muscle myotubes exposed to 1.0 mmol / l palmitate for 16 h. values represent mean se ( n = 512 ) .
differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . *
p < 0.05 , significantly different from the high - fat diet control mice . auc , area under the curve .
metabolic profiling of mice provided further insight with regards to mitochondrial function in obese , insulin - resistant mice , as control - treated dio mice had a significant increase in long - chain acyl carnitine esters versus their lean counterparts ( table 3 ) , indicative of mitochondrial overload and the incomplete oxidation of fatty acids ( 19 ) . however , the accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice was even greater ( table 3 ) .
this suggests that incomplete fatty acid oxidation rates were even more pronounced in the myriocin - treated dio mice , but these animals also had a significant reduction in short - chain acyl carnitine ester content ( table 3 ) , which is consistent with long - chain acyl - coa dehydrogenase inhibition and reduced oxidation of long - chain fatty acids .
metabolic profiling of gastrocnemius muscle from lean and obese mice treated with either vehicle control or myriocin values reported are in pmol / mg protein from n = 6 mice .
differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the lf counterpart .
after 3 weeks of treatment with myriocin , in vivo heat production and ambulatory activity were assessed in our clams apparatus . paralleling our observations with regard to whole - body oxygen consumption rates
, obesity caused a decline in whole - body heat production that was reversed by myriocin treatment ( supplementary fig .
moreover , obesity - induced insulin resistance was associated with reductions in physical activity that were not altered by myriocin treatment ( supplementary fig .
investigation of the lipid metabolite profile in gastrocnemius muscle demonstrated that chronic high - fat feeding increased long - chain acyl - coa , ceramide and dag content , but only a trend to an increase in tag content was observed ( fig .
d ) . treatment with myriocin in obese mice increased gastrocnemius tag content in comparison to their low - fat counterparts , but did not change the dio - associated rise in long - chain acyl - coa and dag content , and as expected , resulted in a dramatic reduction in ceramide content ( fig .
d ) . these results suggest a key role for ceramide in mediating skeletal muscle insulin resistance , and indicate that the other lipid metabolites possibly may not be as important in the insulin - resistance development .
further support for this statement is seen with the positive correlation between ceramide content and the area under the curve during the glucose tolerance test , whereas no correlation was observed with any of the other lipid metabolites ( fig .
interestingly , in a previous study , we showed that mice deficient for malonyl coa decarboxylase ( mcd/ ) are protected from obesity - induced glucose intolerance and insulin resistance , which was associated with a reduction in incomplete fatty acid oxidation rates ( 19 ) . in this study
we show that these same mcd/ mice do not accumulate ceramide in their gastrocnemius muscle after 12 weeks of high - fat feeding ( fig .
6 ) , although they did accumulate other lipid metabolites such as long - chain acyl - coa ( 19 ) .
inhibition of spt1 reduces skeletal muscle ceramide levels with no effect on other lipid metabolites .
d : gastrocnemius triacylglycerol ( tag ) ( a ) , long - chain acyl - coa ( b ) , ceramide ( c ) , and diacylglycerol ( d ) levels in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin .
differences were determined using a two - way anova followed by bonferroni post hoc analysis .
* p < 0.05 , significantly different from the low - fat diet counterpart . p < 0.05 , significantly different from the high - fat diet control mice .
h : correlation between the respective areas under the curve during the glucose tolerance test and ceramide ( e ) , tag ( f ) , long - chain acyl - coa ( g ) , and diacylglycerol ( h ) content of ( n = 1418 ) samples .
malonyl coa decarboxylase - deficient mice ( mcd/ ) do not accumulate skeletal muscle ceramide after 12 weeks of high - fat feeding .
a : area under the curve during a glucose tolerance test after 12 weeks of high - fat feeding in wild - type and mcd/ mice .
b : corresponding gastrocnemius ceramide levels in mcd/ mice after 12 weeks of high - fat feeding .
differences were determined using a two - way anova followed by bonferroni post hoc analysis .
p < 0.05 , significantly different from the high - fat diet wild - type mice . auc , area under the curve . to determine if ceramides may also be involved in genetic forms of insulin resistance and type 2 diabetes , we treated leptin receptor deficient ( db / db ) mice with myriocin to see if we could prevent the progression of insulin resistance in these animals .
we split db / db mice at 6 weeks of age into two groups , and ensured that there were no differences in glucose tolerance before initiating treatment with myriocin ( fig .
both the db / db control and myriocin - treated groups experienced similar body weight increases after 2 weeks of treatment ( data not shown ) , however , although the db / db control group became glucose intolerant , the db / db group treated with myriocin did not ( fig .
fasting blood glucose levels were also significantly lower in the db / db mice treated with myriocin , and although their response to insulin was delayed , myriocin - treated db / db mice demonstrated lower blood glucose levels at nearly all time points during an insulin tolerance test ( fig . 7d
f ) . placing these animals in the clams apparatus yielded a profile similar to that of the dio mice .
the db / db controls had a lower rer in the dark cycle than db/+ lean mice , and had lower whole - body oxygen consumption rates and ambulatory activity , but no change in overall heat production ; interestingly , myriocin treatment of db / db mice did not restore any of these altered parameters in db / db controls , except for a restoration of whole - body oxygen consumption rates during the light cycle ( fig .
examination of the lipid metabolite profile revealed that tag and long - chain acyl - coa levels were elevated in gastrocnemius muscle of db / db controls versus db/+ lean mice , whereas , unexpectedly , dag and ceramide levels were similar between the two groups ( fig .
h ) . myriocin treatment of db / db mice had no effect on tag , long - chain acyl - coa , or dag levels in gastrocnemius muscle versus db / db control mice , but did lead to a dramatic reduction in ceramide levels ( fig .
insulin - stimulated akt and gsk3 phosphorylation were also depressed in db / db control versus db/+ lean mice , but showed an improvement in db / db mice treated with myriocin ( fig .
prevention of insulin resistance in db / db mice via myriocin treatment . a : pretreatment glucose tolerance test ( gtt ) in db / db mice at 6 weeks of age .
c : respective areas under the curve for the post - treatment gtt in db / db mice .
d : insulin tolerance test ( itt ) in db / db mice treated with vehicle control or myriocin .
f : fed and fasted plasma glucose levels in db / db mice treated with vehicle control or myriocin .
differences were determined using either a two - tailed student t test , or a one - way or two - way anova followed by bonferroni post hoc analysis .
* p < 0.05 , significantly different from the db / db control mice . in vivo metabolic parameters , intramyocellular lipid metabolite profile , and insulin signaling in db / db mice treated with myriocin .
rer ( a ) , whole - body oxygen consumption ( b ) , heat production ( c ) , and ambulatory activity ( d ) in db/+ heterozygous mice , and db / db mice treated with vehicle control or myriocin .
gastrocnemius triacylglycerol ( e ) , long - chain acyl - coa ( f ) , diacylglycerol ( g ) , and ceramide levels ( h ) in db/+ heterozygous mice , and db / db mice treated with vehicle control or myriocin .
i : insulin stimulated akt phosphorylation at serine 473 , and ( j ) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of db/+ heterozygous mice and db / db mice treated with vehicle control or myriocin .
differences were determined using either a one - way or two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from the db / db control mice .
as expected , mice fed a high - fat diet for 12 weeks became obese as indicated by a significant increase in weight gain , ( supplementary fig .
d ) . diet - induced insulin - resistant and lean mice were placed in a comprehensive lab animal - monitoring system ( clams ) for whole - body metabolic assessment , which demonstrated a high - fat diet induced shift in fuel preference toward fatty acids as an oxidative energy source , indicated by the large drop in the respiratory exchange ratio ( rer ) ( supplementary fig .
further support for an increase in fatty acid oxidation in obese mice is seen with the increase in gastrocnemius -hydroxyacyl - coa dehydrogenase ( had ) activity ( table 1 ) .
contrary to previous findings ( 16,17 ) , we also report here that obesity induced by chronic high - fat feeding impairs whole - body oxygen consumption rates ( supplementary fig . 2c and d ) .
-hydroxyacyl - coa dehydrogenase ( had ) activity in gastrocnemius muscle of lean and obese mice treated with vehicle control or myriocin values reported are mol / g wet weight / min of n = 5 mice .
differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart .
after 12 weeks of low- or high - fat diet , mice were treated with either myriocin ( 0.5 mg / kg every other day ) or vehicle control .
after 2 weeks of treatment , we demonstrated that inhibition of spt1 with myriocin reverses diet - induced insulin resistance , as determined by glucose tolerance and insulin tolerance testing ( fig .
if these protective effects took place at the skeletal muscle level , a group of animals were killed at 30 min after insulin injection during the insulin tolerance test , and muscles were excised and harvested for immunoblot analysis of the insulin - signaling pathway .
we demonstrate that insulin stimulation of both akt and glycogen synthase kinase 3 ( gsk3 ) phosphorylation were significantly improved in the gastrocnemius muscle of obese mice treated with myriocin ( fig .
phosphorylation of 5amp activated protein kinase ( ampk ) , another key signaling molecule regulating glucose metabolism , did not differ in gastrocnemius muscle of control and myriocin - treated obese mice ( data not shown ) .
inhibition of serine palmitoyl transferase 1 ( spt1 ) reverses high - fat diet induced insulin resistance and improves insulin signaling . a : glucose tolerance test in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin .
c : insulin tolerance test in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin .
e : insulin - stimulated akt phosphorylation at serine 473 , and ( f ) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of obese insulin - resistant mice treated with either vehicle control or myriocin .
values represent mean se ( n = 812 for a d ; n = 4 for e and f ) .
differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from all other groups .
myriocin treatment was without effect on food intake , body weight , and plasma insulin levels , but did reduce both postprandial and fasted plasma glucose levels in obese , insulin - resistant mice ( table 2 ) . although fasting plasma insulin levels did not differ between diet - induced obesity ( dio ) mice treated with vehicle - control or myriocin , more sophisticated studies monitoring the changes in plasma insulin in response to a meal tolerance test in the obese jcr : la cp rat illustrate a significant improvement in plasma insulin control after treatment with the spt1 inhibitor , l - cycloserine ( supplementary fig .
3 ) . interestingly , indirect calorimetry revealed that the improved insulin sensitivity in dio mice treated with myriocin was not associated with a decrease in fatty acid oxidation and an increase in carbohydrate oxidation , as similar rer values were observed between the dio control and myriocin - treated animals ( fig .
2 ) . effect of myriocin on body and tissue weight , and plasma glucose and insulin levels in lean and obese mice values reported are from n = 511 mice .
differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart ; significantly different from high - fat control .
twenty - four - hour ( a ) , dark cycle ( b ) , and light cycle respiratory exchange ratio ( c ) in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin .
differences were determined using a two - way anova followed by a bonferroni post hoc analysis . *
p < 0.05 , significantly different from the low - fat diet counterpart . in a parallel series of experiments , after 12 weeks of high - fat feeding , mice were treated with either the spt1 inhibitor , l - cycloserine ( 25 mg / kg every other day ) or vehicle control .
although not as dramatic as the results observed with myriocin , at 2 weeks after treatment , we report improvements in glucose and insulin tolerance in mice treated with l - cycloserine ( supplementary fig .
obese , insulin - resistant mice were run on an exercise treadmill to determine exercise capacity . as expected , obese mice showed a dramatic reduction in both their treadmill time and distance when compared with their lean counterparts ( fig .
interestingly , treatment of obese mice for 2 weeks with myriocin reversed this reduction in exercise capacity ( fig . 3a and b ) .
this improvement in exercise capacity observed in obese mice treated with myriocin can be explained by enhanced whole - body oxygen consumption rates compared with their control counterparts ( fig .
c ) . in addition , we observed greater citrate synthase activity in gastrocnemius muscle of obese mice treated with myriocin ( fig .
protein expression of peroxisome proliferator - activated receptor- coactivator-1 ( pgc1 ) , a transcriptional coactivator that plays a key role in regulating a number of genes involved in energy metabolism ( 18 ) , showed a trend toward a reduction in control - treated dio mice ( p = 0.077 ) that was not apparent in myriocin - treated dio mice ( fig .
furthermore , we also demonstrate that pretreatment with myriocin increases citrate synthase activity in c2c12 myotubes exposed to 1.0 mmol / l palmitate for 16 h ( fig .
these observations illustrate improvements in mitochondrial function , possibly explaining why exercise capacity and whole - body oxygen consumption rates were enhanced in this group .
time ( a ) and distance ( b ) during an exercise capacity challenge on a running treadmill .
differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart .
myriocin treatment reverses the impairment in whole - body oxygen consumption rates caused by dio .
c : twenty - four hour ( a ) , dark cycle ( b ) , and light cycle ( c ) whole - body oxygen consumption assessment in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin .
d : gastrocnemius muscle citrate synthase activity in vehicle control and myriocin - treated dio mice .
e : pgc1 expression in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin .
f : citrate synthase activity in vehicle control and myriocin - pretreated c2c12 skeletal muscle myotubes exposed to 1.0 mmol / l palmitate for 16 h. values represent mean se ( n = 512 ) .
differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis .
* p < 0.05 , significantly different from the low - fat diet counterpart . p < 0.05 , significantly different from the high - fat diet control mice . auc , area under the curve .
metabolic profiling of mice provided further insight with regards to mitochondrial function in obese , insulin - resistant mice , as control - treated dio mice had a significant increase in long - chain acyl carnitine esters versus their lean counterparts ( table 3 ) , indicative of mitochondrial overload and the incomplete oxidation of fatty acids ( 19 ) . however , the accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice was even greater ( table 3 ) .
this suggests that incomplete fatty acid oxidation rates were even more pronounced in the myriocin - treated dio mice , but these animals also had a significant reduction in short - chain acyl carnitine ester content ( table 3 ) , which is consistent with long - chain acyl - coa dehydrogenase inhibition and reduced oxidation of long - chain fatty acids .
metabolic profiling of gastrocnemius muscle from lean and obese mice treated with either vehicle control or myriocin values reported are in pmol / mg protein from n = 6 mice .
differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the lf counterpart .
after 3 weeks of treatment with myriocin , in vivo heat production and ambulatory activity were assessed in our clams apparatus . paralleling our observations with regard to whole - body oxygen consumption rates
, obesity caused a decline in whole - body heat production that was reversed by myriocin treatment ( supplementary fig .
moreover , obesity - induced insulin resistance was associated with reductions in physical activity that were not altered by myriocin treatment ( supplementary fig .
investigation of the lipid metabolite profile in gastrocnemius muscle demonstrated that chronic high - fat feeding increased long - chain acyl - coa , ceramide and dag content , but only a trend to an increase in tag content was observed ( fig .
d ) . treatment with myriocin in obese mice increased gastrocnemius tag content in comparison to their low - fat counterparts , but did not change the dio - associated rise in long - chain acyl - coa and dag content , and as expected , resulted in a dramatic reduction in ceramide content ( fig .
d ) . these results suggest a key role for ceramide in mediating skeletal muscle insulin resistance , and indicate that the other lipid metabolites possibly may not be as important in the insulin - resistance development .
further support for this statement is seen with the positive correlation between ceramide content and the area under the curve during the glucose tolerance test , whereas no correlation was observed with any of the other lipid metabolites ( fig .
h ) . interestingly , in a previous study , we showed that mice deficient for malonyl coa decarboxylase ( mcd/ ) are protected from obesity - induced glucose intolerance and insulin resistance , which was associated with a reduction in incomplete fatty acid oxidation rates ( 19 ) . in this study
we show that these same mcd/ mice do not accumulate ceramide in their gastrocnemius muscle after 12 weeks of high - fat feeding ( fig .
6 ) , although they did accumulate other lipid metabolites such as long - chain acyl - coa ( 19 ) .
inhibition of spt1 reduces skeletal muscle ceramide levels with no effect on other lipid metabolites .
d : gastrocnemius triacylglycerol ( tag ) ( a ) , long - chain acyl - coa ( b ) , ceramide ( c ) , and diacylglycerol ( d ) levels in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin .
differences were determined using a two - way anova followed by bonferroni post hoc analysis .
* p < 0.05 , significantly different from the low - fat diet counterpart . p < 0.05 , significantly different from the high - fat diet control mice .
h : correlation between the respective areas under the curve during the glucose tolerance test and ceramide ( e ) , tag ( f ) , long - chain acyl - coa ( g ) , and diacylglycerol ( h ) content of ( n = 1418 ) samples .
malonyl coa decarboxylase - deficient mice ( mcd/ ) do not accumulate skeletal muscle ceramide after 12 weeks of high - fat feeding .
a : area under the curve during a glucose tolerance test after 12 weeks of high - fat feeding in wild - type and mcd/ mice .
b : corresponding gastrocnemius ceramide levels in mcd/ mice after 12 weeks of high - fat feeding .
differences were determined using a two - way anova followed by bonferroni post hoc analysis . *
p < 0.05 , significantly different from low - fat diet counterpart . p < 0.05 , significantly different from the high - fat diet wild - type mice .
to determine if ceramides may also be involved in genetic forms of insulin resistance and type 2 diabetes , we treated leptin receptor deficient ( db / db ) mice with myriocin to see if we could prevent the progression of insulin resistance in these animals .
we split db / db mice at 6 weeks of age into two groups , and ensured that there were no differences in glucose tolerance before initiating treatment with myriocin ( fig .
both the db / db control and myriocin - treated groups experienced similar body weight increases after 2 weeks of treatment ( data not shown ) , however , although the db / db control group became glucose intolerant , the db / db group treated with myriocin did not ( fig .
fasting blood glucose levels were also significantly lower in the db / db mice treated with myriocin , and although their response to insulin was delayed , myriocin - treated db / db mice demonstrated lower blood glucose levels at nearly all time points during an insulin tolerance test ( fig .
f ) . placing these animals in the clams apparatus yielded a profile similar to that of the dio mice .
the db / db controls had a lower rer in the dark cycle than db/+ lean mice , and had lower whole - body oxygen consumption rates and ambulatory activity , but no change in overall heat production ; interestingly , myriocin treatment of db / db mice did not restore any of these altered parameters in db / db controls , except for a restoration of whole - body oxygen consumption rates during the light cycle ( fig .
examination of the lipid metabolite profile revealed that tag and long - chain acyl - coa levels were elevated in gastrocnemius muscle of db / db controls versus db/+ lean mice , whereas , unexpectedly , dag and ceramide levels were similar between the two groups ( fig .
h ) . myriocin treatment of db / db mice had no effect on tag , long - chain acyl - coa , or dag levels in gastrocnemius muscle versus db / db control mice , but did lead to a dramatic reduction in ceramide levels ( fig .
insulin - stimulated akt and gsk3 phosphorylation were also depressed in db / db control versus db/+ lean mice , but showed an improvement in db / db mice treated with myriocin ( fig .
prevention of insulin resistance in db / db mice via myriocin treatment . a : pretreatment glucose tolerance test ( gtt ) in db / db mice at 6 weeks of age .
c : respective areas under the curve for the post - treatment gtt in db / db mice .
d : insulin tolerance test ( itt ) in db / db mice treated with vehicle control or myriocin .
f : fed and fasted plasma glucose levels in db / db mice treated with vehicle control or myriocin .
differences were determined using either a two - tailed student t test , or a one - way or two - way anova followed by bonferroni post hoc analysis .
* p < 0.05 , significantly different from the db / db control mice . in vivo metabolic parameters , intramyocellular lipid metabolite profile , and insulin signaling in db / db mice treated with myriocin .
rer ( a ) , whole - body oxygen consumption ( b ) , heat production ( c ) , and ambulatory activity ( d ) in db/+ heterozygous mice , and db / db mice treated with vehicle control or myriocin .
gastrocnemius triacylglycerol ( e ) , long - chain acyl - coa ( f ) , diacylglycerol ( g ) , and ceramide levels ( h ) in db/+ heterozygous mice , and db / db mice treated with vehicle control or myriocin .
i : insulin stimulated akt phosphorylation at serine 473 , and ( j ) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of db/+ heterozygous mice and db / db mice treated with vehicle control or myriocin .
differences were determined using either a one - way or two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from the db / db control mice .
our results show that inhibition of spt1 reduces de novo ceramide synthesis in muscle , which has novel effects on whole - body energy metabolism and is associated with a profound reversal of glucose intolerance and insulin resistance induced by chronic high - fat feeding .
furthermore , we show that these improvements are dissociated from the other lipid metabolites believed to play a role in the development of insulin resistance .
interestingly , obesity - induced insulin resistance in mice is associated with a detriment in aerobic exercise capacity and whole - body oxygen consumption rates , both of which are partially reversed via spt1 inhibition .
previous studies have postulated that skeletal muscle insulin resistance is caused by the intramyocellular cytosolic accumulation of lipid metabolites ( tag , long - chain acyl - coa , dag , ceramide , etc . )
in particular , long - chain acyl - coa and dag have received considerable attention because of their ability to activate the classic / novel protein kinase c signaling cascade , which can phosphorylate insulin receptor substrate proteins on serine residues , preventing their activation via the insulin receptor ( 4,5,8,2123 ) .
it is important to note , however , that most ( 95% ) acyl - coa esters are located inside the mitochondria ( 24,25 ) , suggesting that if long - chain acyl - coa accumulation does play a role toward insulin resistance development , it is possible that mitochondrial , as opposed to cytosolic long - chain acyl - coa , is the primary contributor .
although tag has been shown in numerous studies to be elevated in muscle in association with the development of insulin resistance , recent studies have shown that tag may actually serve as a buffer , protecting the muscle against the accumulation of the more reactive lipid metabolite species ( 10,11 ) . in regards to ceramide , data are mixed with its role in insulin resistance development , because in some studies , ceramide accumulation is not evident in muscle ( 5,26 ) , and in other studies where accumulation does occur , the relative increase in the ceramide pool is not that large ( 12,27 ) .
( 12 ) has shed some light on this issue , as they demonstrated that ceramide accumulation in muscle is dependent on the type of diet fed to the animals . in
particular , saturated fatty acids drive de novo ceramide synthesis in muscle via spt1 , whereas unsaturated fatty acids cause insulin resistance via other mechanisms ( 12 ) .
such findings may potentially explain why ceramide accumulation is not observed in studies of insulin resistance where the model employed is a lipid infusion that consists primarily of unsaturated fatty acids ( 22 ) .
furthermore , holland et al . ( 12 ) showed in their study that preventing de novo synthesis of ceramide via spt1 inhibition with myriocin prevented the development of glucose intolerance in obese zucker rats , and prevented the palmitate - induced inhibition of insulin - stimulated 2-deoxyglucose uptake in isolated soleus muscle .
( 28 ) also reported positive findings with myriocin treatment in leptin - deficient and dio mice , providing further support that ceramide plays a key role in the development of insulin resistance .
interestingly , these authors also observed a weight loss effect due to myriocin treatment that we did not observe in our studies .
however , the authors in this study used a much longer treatment than ours ( 8 vs. 4 weeks ) , and noted that they did not observe a weight loss effect until later in the treatment period .
furthermore , 3 weeks of myriocin treatment in dio mice improved hyperglycemia and whole - body oxygen consumption rates in their mice , despite no change in body weight compared with control - treated dio mice , which is consistent with our results in dio mice treated with myriocin for 2 weeks .
yang et al . also observed a dramatic reduction in hepatic steatosis that is consistent with our observations in regards to hepatic tag content .
our study adds further support to the studies examining the role of ceramide in mediating insulin resistance ( 12,28 ) by illustrating the potential for targeting spt1 as a treatment against insulin resistance .
. moreover , by examining other lipid metabolites such as tag , dag , long - chain acyl - coa , and acyl carnitine content in skeletal muscle , we are able to discern important differences with regard to the relative importance of each metabolite toward the development of skeletal mucle insulin resistance .
importantly , reductions in skeletal muscle ceramide accumulation may represent a potential explanation for the exercise paradox observed in humans .
dube et al . ( 15 ) showed that obese , insulin - resistant men placed on an aerobic exercise training regime have elevated intramyocellular lipid and tag stores . however , marked reductions in muscle ceramide levels are observed , which may explain the enhanced insulin sensitivity of these men .
moreover , bruce et al . ( 14 ) showed that the improved insulin sensitivity observed with exercise training in humans is associated with a drop in muscle ceramide levels , and in particular , the saturated species .
( 29 ) showed that exercise training of rats leads to a dramatic drop in the saturated species of ceramide in muscle , which is associated with an enhanced 2-deoxyglucose uptake .
in addition , mice overexpressing diacylglycerol acyl transferase in muscle are protected from high - fat - diet
induced insulin resistance and palmitate inhibition of 2-deoxyglucose uptake in isolated muscle , both of which are associated with an elevation of muscle tag and drop in ceramide levels ( 10 ) .
our results support these studies , as we show that obese , insulin - resistant mice treated with myriocin had significant increases in intramyocellular tag , long - chain acyl - coa , and dag , but a dramatic drop in ceramide content .
moreover , we observed a positive correlation with ceramide content and glucose intolerance , but not with any of the other lipid metabolites .
we believe that with this finding , in the setting of obesity , that ceramide may be more vital to the development of skeletal muscle insulin resistance than the other lipid metabolites .
support for this statement is also evident in culture models of ceramide accumulation , whereby inhibition of spt1 was able to prevent palmitate - induced insulin resistance in both human and rat l6 myotubes , despite elevated tag and dag levels ( 11,13 ) .
furthermore , a recent study in humans demonstrated that insulin resistant muscle is associated with elevated ceramide content , but no change in dag content ( 30 ) .
nonetheless , it is also important to note that our measurement of dag assessed total cellular levels of dag , and it is possible that differences in plasma membrane dag were significantly reduced via myriocin treatment . because dag at the membrane is believed to be the specific dag pool responsible for mediating skeletal muscle insulin resistance ( 3 ) , it will be important for future studies to investigate this in more detail .
one of the most surprising findings of this study was that chronic high - fat feeding resulted in a dramatic decline in whole - body oxygen consumption rates .
the majority of studies that have examined the effect of high - fat feeding on whole - body oxygen consumption rates via use of the clams apparatus have reported elevations in oxygen consumption rates ( 16,17 ) .
although the differences between these studies and ours could be due to the duration or composition of the diet , we propose two possible explanations for this observation of ours .
first , it has been reported that obesity - induced insulin resistance causes mitochondrial dysfunction that results from an impairment of fatty acid oxidative capacity ( 58 ) . although it may be possible that our model of insulin resistance is inducing mitochondrial dysfunction , it is highly unlikely due to impairments in muscle fatty acid oxidative capacity , as the rer values in obese mice reported in this study are very close to 0.7 , indicating that these animals have no trouble utilizing fat as an energy source .
nonetheless , other factors , such as mitochondrial content , protein expression of electron transport chain ( etc ) complexes , or activity of these complexes , may account for potential mitochondrial dysfunction and the subsequent impairment of oxygen consumption rates observed in obese mice ( 31,32 ) .
however , we did not observe differences in protein expression of cytochrome c of the etc in any group ( data not shown ) .
second , and just as relevant to the findings of this study , is that obesity - induced insulin resistance has been associated with elevated rates of incomplete fatty acid oxidation , which can arise when rates of fatty acid oxidation are disconnected from tca cycle activity ( 19,33,34 ) .
this disconnect arises due to the sedentary nature of obese individuals and animals , thus there is no demand for the tca cycle to upregulate its activity to deal with the increased fatty acid supply that is being utilized as an energy source ( 19,33,34 ) .
if the tca cycle is unable to accommodate the increasing acetyl coa coming from fatty acid oxidation , reducing equivalents such as nadh and fadh2 would not donate their electrons to the complexes of the etc , accounting for the reduction in oxygen consumption rates .
our observation of increased accumulation of long - chain acyl carnitine esters in the muscle of dio mice is thus consistent with elevated rates of incomplete fatty acid oxidation .
in contrast , there was an even greater accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice , which at first glance would suggest even greater rates of incomplete fatty acid oxidation in these animals .
however , myriocin - treated dio mice actually had a significant reduction in the content of a number of short - chain acyl carnitine esters , and this , in combination with the rise of long - chain acyl carnitine esters , is suggestive of long - chain acyl - coa dehydrogenase and subsequent long - chain fatty acid oxidation inhibition ( 35 ) .
another piece of indirect support for fatty acid oxidation inhibition with myriocin treatment in dio mice is the observation that tag accumulated in the muscle of these animals versus their lean counterparts , but not in control - treated dio mice versus their lean counterparts .
a reduction in fatty acid oxidation - derived nadh would decrease nadh / nadph oxidase activity and subsequent superoxide production in myriocin - treated dio mice , which would contribute toward their improved mitochondrial function .
this improvement in mitochondrial function , coupled together with improvements in glucose metabolism and glucose - derived acetyl coa production for the tca cycle , may contribute to the greater oxygen consumption rates in these animals .
obesity - induced decrements in pgc1 protein expression might also explain impairments in mitochondrial function ( 34,36,37 ) , and although not significant , we observed a trend toward a reduction in gastrocnemius pgc1 protein expression in control - treated dio mice ( p = 0.077 ) that was not evident in myriocin - treated dio mice .
interestingly , citrulline levels were increased in myriocin - treated dio mice versus their control counterparts ( supplementary fig .
a previous study in humans showed that supplementation of citrulline enhances aerobic oxidative metabolism ( 38 ) , supporting our findings of increased whole - body oxygen consumption rates and greater exercise time in myriocin - treated dio mice .
how myriocin and subsequent spt1 inhibition would influence skeletal muscle citrulline levels is currently unknown , but is undoubtedly an intriguing avenue for future investigation .
in addition , we have previously shown that mcd/ mice ( a genetic model of fatty acid oxidation deficiency ) are protected from obesity - induced insulin resistance .
interestingly , we show in this study that these exact same animals do not accumulate ceramide in their muscle after 12 weeks of high - fat feeding , leading to the very intriguing possibility that intramyocellular ceramide accumulation is linked to the mitochondrial dysfunction and enhanced skeletal muscle fatty acid oxidation rates observed in insulin resistance .
a limitation with our interpretation of whole - body oxygen consumption rates is that , unlike human studies , we were unable to normalize our oxygen consumption rates to lean body mass .
it is entirely possible that whole - body oxygen consumption rates were simply lower in dio mice because of a significant increase in overall adiposity , due to fat mass having a lower metabolic rate than lean body mass .
however , the fact that adiposity and body weight were similar between myriocin- and control - treated dio mice suggests that this would not be a contributing factor to the higher oxygen consumption rates observed in the myriocin - treated dio mice .
although we believe that the changes accounting for the greater oxygen consumption rates in myriocin - treated dio mice primarily reflect the muscle , we can not ignore possible contributions from changes in other peripheral tissues , such as brown adipose tissue and uncoupling protein activity .
the beneficial effects mediated by inhibition of spt1 and prevention of de novo ceramide synthesis could also arise from liver effects in our animals .
regardless , we did not observe increases in hepatic ceramide content after diet - induced obesity , and myriocin treatment had no effect on insulin - stimulated akt and gsk3 phosphorylation in obese mice versus their respective controls ( supplementary fig .
, recent studies have also shown that high - fat feeding does not increase ceramide content in the liver ( 39 ) , and increases in hepatic ceramide content via genetic overexpression of either dgat1 or dgat2 does not result in any type of insulin resistance or inflammation ( 40 ) .
moreover , we reported no difference during a pyruvate challenge of fasted , obese , control- or myriocin - treated mice ( supplementary fig . 8) , suggesting that gluconeogenic capacity was not different between the two groups and that the liver likely does not play a key role with the improved insulin sensitivity observed in myriocin - treated mice .
regardless , we can not entirely rule out the possibility that the liver plays a role with the benefit observed during myriocin treatment , as the dio - associated rise in hepatic tag content was reversed via myriocin treatment , and thus it will be important for future studies to delineate the role of hepatic spt1 in greater depth .
finally , chronic low - grade inflammation has been shown in a number of studies to play a role in causing obesity - induced insulin resistance ( 4143 ) .
inflammatory and stress kinases , such as p38 mapk and jnk , have been proposed to be downstream mediators of this inflammatory effect , as inhibitors of both kinases are able to prevent high - fat diet induced insulin resistance ( 9,4446 ) . unexpectedly ,
the phosphorylation status of both p38 mapk and jnk was not altered by dio , nor was it altered by myriocin treatment ( supplementary fig .
9 ) , suggesting that inflammation may not play as vital a role in our model of insulin resistance .
it may also be possible that inflammation in our model is mediated by some other kinase , such as ikk ( 47,48 ) . with regard to the findings in db / db mice ,
we report very similar findings to what we observed in the obesity - induced insulin - resistant mice , and that treatment with myriocin also yielded a very similar beneficial profile .
interestingly , gastrocnemius ceramide levels , although reduced in myriocin - treated db / db mice , did not differ between db/+ lean and db / db control mice .
this suggests , at least in this model , that perhaps ceramide metabolites , such as glucosylceramide , are more important in mediating skeletal muscle insulin resistance than ceramide itself ( 49 ) .
furthermore , the ceramide pool is under a dynamic process of synthesis and degradation ( 9 ) , and although de novo synthesis of ceramide may be increased in these animals , a simultaneous increase in ceramide degradation would mask out any noticeable change . in summary , we show that ceramide accumulation in skeletal muscle plays a key role during obesity - induced insulin resistance , whereas the other lipid metabolites , such as tag , long - chain acyl - coa , and dag , may not be as vital .
importantly , inhibition of de novo ceramide synthesis has novel effects on whole - body energy metabolism and is sufficient to reverse obesity - induced whole - body glucose intolerance and insulin resistance .
furthermore , whole - body oxygen consumption rates and exercise capacity in obese mice are improved via inhibition of de novo ceramide synthesis .
last , our finding that muscle ceramide levels are not elevated in db / db mice , but that inhibition of de novo ceramide synthesis still prevents their development of insulin resistance , suggests the possibility that ceramide metabolites may also play a role in the progression of this disease . | objectiveit has been proposed that skeletal muscle insulin resistance arises from the accumulation of intramyocellular lipid metabolites that impede insulin signaling , including diacylglycerol and ceramide .
we determined the role of de novo ceramide synthesis in mediating muscle insulin resistance.research design and methodsmice were subjected to 12 weeks of diet - induced obesity ( dio ) , and then treated for 4 weeks with myriocin , an inhibitor of serine palmitoyl transferase-1 ( spt1 ) , the rate - limiting enzyme of de novo ceramide synthesis.resultsafter 12 weeks of dio , c57bl/6 mice demonstrated a doubling in gastrocnemius ceramide content , which was completely reversed ( 141.5 15.8 vs. 94.6 10.2 nmol / g dry wt ) via treatment with myriocin , whereas hepatic ceramide content was unaffected by dio .
interestingly , myriocin treatment did not alter the dio - associated increase in gastrocnemius diacyglycerol content , and the only correlation observed between lipid metabolite accumulation and glucose intolerance occurred with ceramide ( r = 0.61 ) .
dio mice treated with myriocin showed a complete reversal of glucose intolerance and insulin resistance which was associated with enhanced insulin - stimulated akt and glycogen synthase kinase 3 phosphorylation .
furthermore , myriocin treatment also decreased intramyocellular ceramide content and prevented insulin resistance development in db / db mice . finally , myriocin - treated dio mice displayed enhanced oxygen consumption rates ( 3,041 124 vs. 2,407 124
ml / kg / h ) versus their control counterparts.conclusionsour results demonstrate that the intramyocellular accumulation of ceramide correlates strongly with the development of insulin resistance , and suggests that inhibition of spt1 is a potentially promising target for the treatment of insulin resistance . | RESEARCH DESIGN AND METHODS
RESULTS
Chronic high-fat feeding results in dramatic weight gain, whole-body insulin resistance, and altered substrate preference.
Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 reverses diet-induced insulin resistance.
Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 reverses diet-induced impairments in exercise capacity, which coincide with a restoration of whole-body oxygen consumption rates and inhibition of fatty acid oxidation.
Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 reverses diet-induced impairments on heat production with no effect on animal activity.
Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 has no effect on the obesity-associated increase of long-chain acyl-CoA and DAG accumulation in muscle, but significantly elevates TAG levels.
Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 prevents the development of insulin resistance in leptin receptor deficient type 2 diabetes
DISCUSSION
Supplementary Material |
PMC4989426 | . they can be caused by mutations in nuclear genes either relating to mitochondrial components or mitochondrial dna ( mtdna ) .
the prevalence of mitochondrial diseases due to mutations in mtdna and related nuclear genes is estimated to be 1 in 5000 .
mutations in mtdna are much higher than those in the related nuclear genes , probably because that mitochondrial genome has been exposed in reactive oxygen species(ros ) and lacked protection structure . in 1990 , the adenine to guanine transition at the mtdna position of 3243 encoding trna was found to be the molecular basis for mitochondrial encephalomyopathy with lactic acidosis and stroke - like episodes ( melas ) .
epidemiological studies showed that m.3243a > g mutation is the most frequent pathogenic mutation in mtdna .
the prevalence of m.3243a > g mutation in mtdna is 3.5/100,000 adults in the north east of england and approximately 1/6000 in an adult population in finland .
seizures , encephalopathy , and stroke - like episodes were found in about 80% patients with melas , and short stature , deafness , cognitive impairment , exercise intolerance , migraines , depression , cardiomyopathy , cardiac conduction defects , and diabetes mellitus were also found in some melas cases . here , we summarized the clinical spectrum of m.3243a > g mutation in chinese pediatric patients , to define the common clinical manifestations and study the correlation between heteroplasmic degree of the mutation and clinical severity of the disease .
clinical data of 100 chinese pediatric patients who were first diagnosed as mitochondrial diseases through gene test for m.3243a > g mutation in peking university first hospital from 2007 to 2013 were retrospectively reviewed .
this study was approved by the medical ethics committee of peking university first hospital ( no .
a total of ten clinical characteristics including vision impairment , hearing loss , encephalopathy , myopathy , and gastrointestinal disturbances were collected from these patients .
short stature was defined as the body height less than two standard deviations below the mean height of normal children .
the peripheral blood samples were collected from these patients when they were first screened for the gene mutation .
detection of m.3243a > g mutation ratio was performed by polymerase chain reaction ( pcr)-restriction fragment length polymorphism method .
the pcr product was digested with restriction enzyme apa i , and then separated in 2% agarose gel .
the patients were divided into three groups based on mutation ratio ( low level : 030% ; middle level : 3160% ; and high level : 61100% ) .
age and mutation ratio inconsistent with gaussian distribution were presented as median ( q1 , q3 ) .
correlation between m.3243a > g mutation ratio and age was evaluated with spearman 's rank correlation method .
the differences in clinical symptom frequency of patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test .
clinical data of 100 chinese pediatric patients who were first diagnosed as mitochondrial diseases through gene test for m.3243a > g mutation in peking university first hospital from 2007 to 2013 were retrospectively reviewed .
this study was approved by the medical ethics committee of peking university first hospital ( no .
a total of ten clinical characteristics including vision impairment , hearing loss , encephalopathy , myopathy , and gastrointestinal disturbances were collected from these patients .
short stature was defined as the body height less than two standard deviations below the mean height of normal children .
the peripheral blood samples were collected from these patients when they were first screened for the gene mutation .
detection of m.3243a > g mutation ratio was performed by polymerase chain reaction ( pcr)-restriction fragment length polymorphism method .
the pcr product was digested with restriction enzyme apa i , and then separated in 2% agarose gel .
the patients were divided into three groups based on mutation ratio ( low level : 030% ; middle level : 3160% ; and high level : 61100% ) .
age and mutation ratio inconsistent with gaussian distribution were presented as median ( q1 , q3 ) .
correlation between m.3243a > g mutation ratio and age was evaluated with spearman 's rank correlation method .
the differences in clinical symptom frequency of patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test .
the age of the diagnosis of mitochondrial disease ranged from 2 months to 18 years , with the median age of 9 years ( 5.8 years , 12.0 years ) .
the m.3243a > g mutation ratio varied from 5% to 93% , with a median level of 44% ( 36% , 54% ) .
there was no significant difference in m.3243a > g mutation ratio between males and females ( t = 0.691 , p = 0.491 ) .
patients at the onset had one or more symptoms , including seizures ( 54% ) , muscle weakness ( 29% ) , headache complicated with vomiting ( 25% ) , decreased vision ( 18% ) , hearing loss ( 10% ) , impaired verbal communication ( 6% ) , and heart preexcitation syndrome ( 5% ) .
the most prevalent symptom of these patients was seizures ( 76% ) , followed by short stature ( 73% ) , elevated plasma lactate ( 70% ) , abnormal magnetic resonance imaging / computed tomography ( mri / ct ) changes ( 68% ) , vomiting ( 55% ) , decreased vision ( 52% ) , headache ( 50% ) , and muscle weakness ( 48% ) .
most of the patients were multisymptomatic , only two patients had one symptom , and five patients manifested two symptoms .
seizures were present in 76 patients ( 76% ) , of which 13 patients had stroke during or shortly after seizures .
recurrent headaches were found in 50 patients ( 50% ) , of which most were complicated with vomiting and/or vision loss .
sixty - eight patients ( 68% ) were found to have mri / ct abnormalities , including abnormal asymmetric signals in occipital area ( 51/68 , 75% ) , temporal area ( 21/68 , 31% ) , parietal area ( 20/68 , 29% ) , bilateral basal ganglia calcification ( 16/68 , 24% ) , cerebral atrophy ( 10/68 , 15% ) , and thalamus and brainstem lesions ( 5/68 , 7% ) .
slurred speech was present in 16 patients , of whom 3 had progressively worsening speech , and 2 had delayed speech development ( at 1.5 and 3.0 years of age ) .
seventy - three patients ( 73% ) had a short stature and 69 patients ( 69% ) had a weight loss .
fifty - five patients ( 55% ) had experienced recurrent vomiting and 38 patients ( 38% ) had diarrhea or / and constipation .
plasma lactate ranged from 1.4 to 19.0 mmol / l ( normal range : 0.72.0 mmol / l ) .
elevated plasma lactate was detected in 70 patients ( 70% ) , of which 90% had a plasma lactate > 5 mmol / l .
vision impairment was found in 52 patients ( 52% ) , of which 33 experienced blurred vision and 16 had decreased visual acuity .
reduced muscle strength was reported in 48 patients ( 48% ) , of which 31 had minimal muscle weakness in upper limbs and shoulders and 17 manifested muscle weakness in lower limbs , and 2 had walk difficulties .
thirty - seven patients ( 37% ) complained of difficulties in maintaining stability , of them 18 experienced frequent tumbling , and 38 patients ( 38% ) had compromised exercise tolerance , which appeared during running or going upstairs in most cases and during walk on flat places in only two cases .
heart diseases were detected in 35 patients ( 35% ) , of which 17 were found to have abnormal electrocardiogram such as st - t changes and arrhythmias . left ventricular hypertrophy was found in eight patients , right ventricular hypertrophy in five patients , and preexcitation syndrome in five patients .
twenty - one patients ( 21% ) had hearing impairment , presenting tinnitus or hearing loss ( mild deafness in 14 patients , moderate deafness in 5 patients , and severe deafness in 2 patients ) .
the m.3243a > g mutation ratio in peripheral leukocytes was determined in all the patients , and 32% of them had a mutation ratio above 50% .
the relationship between m.3243a > g mutation ratio and onset age was analyzed by spearman 's rank correlation method , which showed that m.3243a > g mutation ratio was correlated negatively with onset age [ r = 0.470 , p < 0.001 ; figure 1 ] .
correlation between m.3243a > g mutation ratio in peripheral leukocytes and onset age ( r=0.470 , n=100 ) .
the differences in clinical symptom frequency among patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test [ table 1 ] .
there were significant differences in the frequencies of hearing loss , decreased vision , myopathy , and gastrointestinal disturbance among three groups .
however , patients with a low or middle level of m.3243a > g mutation ratio were more likely to suffer from hearing loss , decreased vision , and gastrointestinal disturbance than high level group .
analysis of clinical symptom frequency in different distributions of 3243a > g mutation ratio , n low level : a mutation ratio range 030% ; middle level : a mutation ratio range 3160% ; high level : a mutation ratio range 61100% ; mri : magnetic resonance imaging ; ct : computed tomography .
patients at the onset had one or more symptoms , including seizures ( 54% ) , muscle weakness ( 29% ) , headache complicated with vomiting ( 25% ) , decreased vision ( 18% ) , hearing loss ( 10% ) , impaired verbal communication ( 6% ) , and heart preexcitation syndrome ( 5% ) .
the most prevalent symptom of these patients was seizures ( 76% ) , followed by short stature ( 73% ) , elevated plasma lactate ( 70% ) , abnormal magnetic resonance imaging / computed tomography ( mri / ct ) changes ( 68% ) , vomiting ( 55% ) , decreased vision ( 52% ) , headache ( 50% ) , and muscle weakness ( 48% ) .
most of the patients were multisymptomatic , only two patients had one symptom , and five patients manifested two symptoms .
seizures were present in 76 patients ( 76% ) , of which 13 patients had stroke during or shortly after seizures .
recurrent headaches were found in 50 patients ( 50% ) , of which most were complicated with vomiting and/or vision loss .
sixty - eight patients ( 68% ) were found to have mri / ct abnormalities , including abnormal asymmetric signals in occipital area ( 51/68 , 75% ) , temporal area ( 21/68 , 31% ) , parietal area ( 20/68 , 29% ) , bilateral basal ganglia calcification ( 16/68 , 24% ) , cerebral atrophy ( 10/68 , 15% ) , and thalamus and brainstem lesions ( 5/68 , 7% ) .
slurred speech was present in 16 patients , of whom 3 had progressively worsening speech , and 2 had delayed speech development ( at 1.5 and 3.0 years of age ) .
seventy - three patients ( 73% ) had a short stature and 69 patients ( 69% ) had a weight loss .
fifty - five patients ( 55% ) had experienced recurrent vomiting and 38 patients ( 38% ) had diarrhea or / and constipation .
plasma lactate ranged from 1.4 to 19.0 mmol / l ( normal range : 0.72.0 mmol / l ) .
elevated plasma lactate was detected in 70 patients ( 70% ) , of which 90% had a plasma lactate > 5 mmol / l .
vision impairment was found in 52 patients ( 52% ) , of which 33 experienced blurred vision and 16 had decreased visual acuity .
reduced muscle strength was reported in 48 patients ( 48% ) , of which 31 had minimal muscle weakness in upper limbs and shoulders and 17 manifested muscle weakness in lower limbs , and 2 had walk difficulties .
thirty - seven patients ( 37% ) complained of difficulties in maintaining stability , of them 18 experienced frequent tumbling , and 38 patients ( 38% ) had compromised exercise tolerance , which appeared during running or going upstairs in most cases and during walk on flat places in only two cases .
heart diseases were detected in 35 patients ( 35% ) , of which 17 were found to have abnormal electrocardiogram such as st - t changes and arrhythmias .
left ventricular hypertrophy was found in eight patients , right ventricular hypertrophy in five patients , and preexcitation syndrome in five patients .
twenty - one patients ( 21% ) had hearing impairment , presenting tinnitus or hearing loss ( mild deafness in 14 patients , moderate deafness in 5 patients , and severe deafness in 2 patients ) .
the m.3243a > g mutation ratio in peripheral leukocytes was determined in all the patients , and 32% of them had a mutation ratio above 50% .
the relationship between m.3243a > g mutation ratio and onset age was analyzed by spearman 's rank correlation method , which showed that m.3243a > g mutation ratio was correlated negatively with onset age [ r = 0.470 , p < 0.001 ; figure 1 ] .
correlation between m.3243a > g mutation ratio in peripheral leukocytes and onset age ( r=0.470 , n=100 ) .
the differences in clinical symptom frequency among patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test [ table 1 ] .
there were significant differences in the frequencies of hearing loss , decreased vision , myopathy , and gastrointestinal disturbance among three groups .
however , patients with a low or middle level of m.3243a > g mutation ratio were more likely to suffer from hearing loss , decreased vision , and gastrointestinal disturbance than high level group .
analysis of clinical symptom frequency in different distributions of 3243a > g mutation ratio , n low level : a mutation ratio range 030% ; middle level : a mutation ratio range 3160% ; high level : a mutation ratio range 61100% ; mri : magnetic resonance imaging ; ct : computed tomography .
the m.3243a > g mutation has been shown to lead to reduced levels of the trna , decrease in aminoacylation , and absence of the normal modification with 5-taurinomethyl group at the wobble base .
mitochondrial disease caused by this mutation may result from the reduction of mtdna - encoded proteins and oxidative phosphorylation activity in mitochondrial translation .
several clinical syndromes including melas , myoclonic epilepsy with ragged - red fibers , chronic progressive external ophthalmoplegia , and leigh 's syndrome may associate with m.3243a > g mutation .
patients with m.3243a > g mutation but without clinical symptoms are not uncommon . to define the spectrum of clinical manifestations due to m.3243a > g mutation
, we retrospectively reviewed clinical data of 100 chinese pediatric patients with m.3243a > g mutation regardless of their clinical presentations .
central nervous system ( cns ) is frequently involved in mitochondrial diseases because of its higher energy demand .
seizures may result from neuronal energy depletion , oxidative stress , impaired calcium signaling , immune disturbances , and deficiencies of vitamins , cofactors , and amino acids . in this study ,
the prevalence of seizures was as high as 76% , and was nearly 100% in our previous study , similar to the prevalence of 70.5% in a report of pediatric mitochondrial disease from taiwan , china .
cns symptoms are , therefore , more common and severe in children than adults . the prevalence of seizures ranged from 9% to 20% in adults . in melas patients , recurrent stroke - like episodes were more frequent in those with m.3243a > g mutation than those without the mutation .
we found stroke - like episodes in 22% patients , higher than the prevalence from patients with mitochondrial mutations other than m.3243a > g mutation .
most of our patients showed abnormal brain image findings were probably not due to vascular injuries .
bilateral basal ganglia were the most vulnerable site in this disease , followed by temporal , parietal , and occipital area , similar to the previous report .
chae et al . also observed the tendency that basal ganglia were frequently involved in patients without m.3243a > g mutation .
vomiting was more frequent than constipation or / and diarrhea , and 68% vomiting cases were complicated with headaches . a higher prevalence ( 42% ) of these symptoms
therefore , mitochondrial diseases should be considered clinically in children with unexplained vomiting and headaches .
the prevalence of cardiac involvement was highly variable in patients with mtdna mutations , ranging from 17% to 40% .
in this study , 35% patients exhibited cardiac involvement with the main manifestations of abnormal electrocardiogram ( 17% ) and left ventricular hypertrophy ( 8% ) .
the prevalence of left ventricular hypertrophy was less in our patients than the patients with m.3243a > g mutation ( 56% ) reported by majamaa - voltti et al .
pediatric patients may be the reason of that lower prevalence of cardiac involvement was present in this study . for patients suspected of mitochondrial disease , routine electrocardiogram , and ultrasonocardiography
the m.3243a > g mutation ratio in peripheral leukocytes was negatively correlated with patients onset age .
myopathy was frequently seen in patients with high level of m.3243a > g mutation ratio .
however , patients with a low or middle level of m.3243a > g mutation ratio were more likely to suffer from hearing loss , decreased vision , and gastrointestinal disturbance . in conclusion , this study summarized the common symptoms and onset symptoms in a large cohort of chinese pediatric patients with m.3243a > g mutation . in view of the fact that the diagnosis of 66% of patients was delayed an average of 2 years
, we suggested that examination of m.3243a > g mutation in mtdna should be considered in children with one or more of the symptoms including seizures , short stature , muscle weakness , headache complicated with vomiting , decreased vision , and hearing loss .
although the mutation ratio in blood sample is an available test for diagnosis of mitochondrial disease , the m.3243a > g mutation ratio is usually higher in muscle and urine sample .
therefore , the mutation ratio in other tissues , such as muscle and urine cell , should be included in the future study .
this study was supported by grants from national natural science foundation of china ( no .
this study was supported by grants from national natural science foundation of china ( no . 81271256 and no . | background : mitochondrial diseases are a group of energy metabolic disorders with multisystem involvements .
variable clinical features present a major challenge in pediatric diagnoses .
we summarized the clinical spectrum of m.3243a > g mutation in chinese pediatric patients , to define the common clinical manifestations and study the correlation between heteroplasmic degree of the mutation and clinical severity of the disease.methods:clinical data of one - hundred pediatric patients with symptomatic mitochondrial disease harboring m.3243a > g mutation from 2007 to 2013 were retrospectively reviewed .
detection of m.3243a > g mutation ratio was performed by polymerase chain reaction ( pcr)-restriction fragment length polymorphism .
correlation between m.3243a > g mutation ratio and age was evaluated .
the differences in clinical symptom frequency of patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test.results:sixty-six patients ( 66% ) had suffered a delayed diagnosis for an average of 2 years .
the most frequent symptoms were seizures ( 76% ) , short stature ( 73% ) , elevated plasma lactate ( 70% ) , abnormal magnetic resonance imaging / computed tomography ( mri / ct ) changes ( 68% ) , vomiting ( 55% ) , decreased vision ( 52% ) , headache ( 50% ) , and muscle weakness ( 48% ) .
the mutation ratio was correlated negatively with onset age ( r = 0.470 , p < 0.001 ) .
myopathy was more frequent in patients with a high level of mutation ratio .
however , patients with a low or middle level of m.3243a > g mutation ratio were more likely to suffer hearing loss , decreased vision , and gastrointestinal disturbance than patients with a high level of mutation ratio.conclusions:our study showed that half of chinese pediatric patients with m.3243a > g mutation presented seizures , short stature , abnormal mri / ct changes , elevated plasma lactate , vomiting , and headache .
pediatric patients with these recurrent symptoms should be considered for screening m.3243a > g mutation .
clinical manifestations and laboratory abnormalities should be carefully monitored in patients with this point mutation . | I
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Subjects
Mitochondrial DNA analysis
Statistic analysis
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Clinical features of patients
Correlation between m.3243A>G mutation ratio and onset age
Clinical symptom frequency of patients with different mutation ratio
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Financial support and sponsorship
Conflicts of interest |
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