however a permanent type 1 diabetes - like status can be experimentally induced either by total pancreatectomy or by chemical destruction of pancreatic -cells with streptozotocin ( stz ) [ 1 , 2 ] . both experimental approaches allow for the establishment of chronic hyperglycemia and low endogenous insulin production in nhps , similarly to what it is found in humans with type1 diabetes . in patients an optimal treatment of diabetes involves control of glycemia by insulin administrations under haemoglobin a1c ( hba1c ) monitoring . daily glucose measurements , even if frequent , do not provide accurate measures of long - term average blood glucose concentrations . the best method to assess long - term glycemic control is the measurement of hba1c . hba1c values are important parameters for physicians and quite helpful to adjust the dose of insulin and antidiabetic drugs for a better control of the disease . evidence supporting the translation of hba1c into glycemic control and long - term risk assessment of microvascular complications has been provided by the diabetes control and complications trial ( dcct )   and the united kingdom prospective diabetes study ( upkds ) . the dcct and the ukpds are landmark clinical trials that compared the effect of intensive glucose - lowering therapies with conventional blood glucose control on the long - term risks of complications in patients with type 1 ( dcct )   and type 2 ( ukpds )   diabetes . both of the trials documented that better glycemic control was associated with improved clinical outcome . hba1c values are strongly correlated with blood glucose levels and with the risk of developing complications . this is the reason why we thought it useful to record hba1c as a parameter to monitor diabetes in nhps ( particularly in long - term islet graft recipients ) as in humans . even if species differences should be taken into consideration , testing of novel therapeutic approaches in nhps is one of the best ways to predict possible effects in humans . clinical signs vary , but there is often a gradual progression of the disease even in nhps . traditional tests for the detection of diabetes mellitus include measurement of fasting plasma glucose concentrations , measurement of urine glucose concentrations , oral ( ogtt ) and iv ( ivgtt ) glucose tolerance tests , measurement of urine ketone concentrations , and measurement of fasting plasma insulin concentrations [ 711 ] . diagnostic criteria are ideally based on the risk of developing long - term microvascular complications [ 1114 ] . while nhps are the recipients of choice for testing alternative sources of pancreatic islets , such tests present challenges in these animals . these include difficulty in sample collection , necessity for anesthesia during blood drawing with the potential for drug interactions , multiple confounding factors ( e.g. , activity , duration of food withholding , or diet ) , stress hyperglycemia attributable to restraint ( i.e. , catecholamine release suppressing insulin secretion ) , and lack of established reference ranges [ 1517 ] . objectives of the study reported here were to identify values for measurement of hba1c percentage in blood samples obtained from nhps ( macaca fascicularis ) to determine whether these percentages varied with respect to glycemic control after diabetes induction and insulin treatment . hba1c measurements were also carried out after pig islet transplantation in diabetic nhp recipients in an attempt to assess whether this physiologic variable can be considered a suitable test to monitor glucose metabolism and provide a positive feedback after islet transplantation , particularly in long - term survivors . even if islet xenotransplantation of porcine islets in nhps restores normal blood glucose levels in diabetic recipients , to date no clear long - term effect has been fully demonstrated . glycated hemoglobin percentage can offer a reliable means to determine the establishment of euglycemia after xenotransplantation . a total of 15 male cynomolgus monkeys ( i.e. , macaca fascicularis , three spring scientific , perkaise , pa , usa ) , 24 years old and 2.84.9  kg ( median 3.4  kg ) , were included in this study ; 6 monkeys were nondiabetic , 9 diabetic , and 3 diabetics received islet transplantations . gt - dko pigs ( -1,3-galactosyltransferase double ko pigs ) or hcd46 transgenic pigs   ( revivicor , blacksburg , va , usa ) were used as sources of pancreata for islet transplantation . all animal care procedures were in accordance with the institutional principles of laboratory animal care ( national society for medical research ) and the guide for the care and use of laboratory animals and were approved by the university of pittsburgh animal care and use committee . diabetes was inducted in 9 monkeys with 125150  mg / kg i.v . of zanosar streptozotocin ( sicor pharmaceutics , irvine , ca , usa ) in a single dose . diabetes was confirmed by persistent hyperglycemia ( > 11.1  mmol / l on at least two consecutive readings ) and by the need for insulin to prevent ketosis . ivgtts ( intravenous glucose tolerance test ) and asts ( arginine stimulation test ) were performed 731 days ( median 12 days ) after induction of diabetes . infusion of insulin ( humulin r ; eli lilly , indianapolis , in , usa ) to maintain the blood glucose level < 11.1  mmol / l and to prevent the development of ketosis . blood glucose ( mmol / l ) was measured in whole blood with a portable glucometer ( freestyle ; abbott laboratories , abbott park , il , usa ) . serum levels of primate c - peptide ( nmol / l ) were measured by radioimmunoassay ( linco research , st charles , mo , usa ) using species - specific antibodies . aprotinin 0.05  kiu / l ( trasylol ; bayer pharmaceuticals , west haven , ct , usa ) was added to the serum as a stabilizing agent . to document the metabolic status of each monkey before induction of diabetes , after induction of diabetes , and after islet transplantation until euthanasia , we recorded mean blood glucose ( mmol / l ) , the prevalence of blood glucose readings > 11.1  mmol / l ( % ) , the mean exogenous insulin requirement ( iu  kg  day ) , and mean porcine ( graft ) c - peptide levels . blood samples for hba1c testing were collected fasting every 24 weeks before and after induction of diabetes . immediately after collection of a sample , blood was transferred into a tube containing edta , which was immediately used for measurement of hba1c percentages . for the measurement of specific hba1c , an inhibition of latex agglutination assay is used ( hba1c - specific mouse monoclonal antibody adsorbent onto latex particles , dca vantage analyzer , siemens healthcare diagnostics , deerfield , il , usa ) . an agglutinator ( synthetic polymer containing multiple copies of the immunoreactive portion of hba1c ) causes agglutination of latex coated with hba1c specific mouse monoclonal antibody . this agglutination reaction causes increased scattering of light , which is measured as an increased absorbance at 531  nm . hba1c in whole blood specimens competes for the limited number of antibody - latex binding sites causing an inhibition of agglutination and decreased scattering of light . the decreased scattering is measured as a decrease absorbance at 531  nm . the hba1c concentration the percent hba1c in the sample is then calculated as follows : % hba1c = [ hba1c]/[total hemoglobin ]  100 . after pancreatectomy in the anesthetized donor pig , islet isolation was carried out according to a modification of the method described for human islets , optimized for pigs   that involved low enzyme concentration , low digestion temperature , and minimal mechanical digestion . intraportal injection of islets ( an average of 40,000100,000 islet equivalents / kg body weight ) was carried out under general anesthesia of recipients . continuous insulin infusion was restored if blood glucose was consistently > 11.1  mmol / l . after induction with antithymocyte globulin , immunosuppression was maintained with humanized anti - cd154 monoclonal antibody ( abi 793 , novartis pharma , basel , switzerland ) and mycophenolate mofetil ( roche , nutley , nj , usa ) . anticoagulation / antiaggregation / anti - inflammatory treatment was achieved with heparin or dextran sulfate , prostacyclin ( glaxosmithkline , research triangle park , nc , usa ) and aspirin ; islet graft function was monitored by measuring porcine c - peptide .   a commercially available technical computing program was used for graphic analyses ( graphpad prism 4 for macintosh graphpad software , la jolla , ca , usa ) . suggested criteria for diabetic classification of subjects were derived from other studies [ 7 , 15 ] that involved the use of nhps . a commercially available statistical program was used for statistical analyses ( graphpad prism 4 for macintosh , graphpad software ) . human data obtained from the literature are presented as the range of values or mean of the published data [ 20 , 21 ] . the data for healthy monkeys were compared with the human data from the literature to better characterize similarities and differences , even though the comparisons are limited by the difference in the testing conditions . for a better characterization of differences between monkeys and humans table 1 also reports fasting blood glucose and c - peptide levels comparisons , based on previous reports . mmol / l and was significantly lower than the corresponding values in humans   ( 3.95.6  mmol / l ; p < .05 ) . human c - peptide   values were consistently lower than monkey c - peptide levels ( 0.47 furthermore , hba1c values for nondiabetic healthy monkeys were lower than those in humans , with statistically significant difference ( 4.4      0.1% in monkeys versus 4.99      0.1% in humans , p < .05 ) . the hba1c of nondiabetic monkeys was compared to that of monkeys that were streptozotocin ( stz ) induced , hyperglycemic , and insulin independent . the increase in hba1c levels following diabetes induction confirms also in nhps the notion that chronically high blood glucose affects glycation of hemoglobin ( figure 1(a ) , p < .05 ) . as expected , during the ivgtt the peak of glucose concentration in diabetic monkeys was significantly higher ( p < .05 , 2  min after glucose i.v . thereafter , the glucose levels decreased at a slower rate in diabetic than nondiabetic animals , as shown by the lower kg ( mean 1.44      0.2  mmol  min , p < .001 ) . the c - peptide increase , seen in nondiabetic monkeys , was absent in diabetic monkeys ( figure 1(c ) ) . during the ast in nondiabetic monkeys ( figures 1(d ) and 1(e ) ) , blood glucose remained stable while c - peptide values rose at 2  min and then returned at prestimulus value at 5 minutes . published data show that human acrarg is similar to that in monkeys ; however , the absolute basal and stimulated values are lower in humans than in monkeys ( 2223 ) . in diabetic monkeys during the ast , blood glucose remained stable at approximately 10.8  mmol / l , and c - peptide showed no response ( arcarg0.400.31  nmol / l ) . in summary , after stz treatment , blood glucose levels in monkeys increased above 10 mmol / l , and fasting levels of endogenous c - peptide declined to values corresponding to 1218% of the c - peptide levels before diabetes induction . any residual endogenous c - peptide did not respond to physiological stimuli , as shown by the results of the dynamic tests and by the absence of a correlation between endogenous c - peptide and blood glucose levels . figure 2 compares the blood glucose profile and hba1c levels in two nhps , during their followup after stz and ( in one case ) islet transplantation . figure 2(a ) shows the blood glucose levels trend after stz of monkey m12408 . despite iv insulin treatment blood glucose level tended to increase , and coherently hba1c values ( red dots ) also tended to increase ( from a basal pre - stz value of 4.3% to 8.2% at the end of followup ) . figure 2(b ) shows a second monkey ( m123 - 08 ) blood glucose profile and hba1c values prior to and after islet transplantation . whereas blood glucose control and hba1c increased after stz , as also shown in monkey m124 - 08 , a substantial decrease in blood glucose levels and a drop in the hba1c level from 9.9% to 6.5 . better physiological glycemic control was achieved by the islet transplant and was reflected in the improved hba1c values , testifying to a better management of the disease . in this experiment a total of three monkeys were transplanted with porcine islets in the liver via the portal vein ; following the transplant , all of them experienced improved metabolic control of glucose . improvement was defined by a statistically significant drop in between blood glucose levels ( 8.87       0.19 mmol / l versus 5.28       0.06  mmol / l , p < .0001 ) and insulin dose ( 1.07      0.06  iu  kg  day versus 0.04      0.01  iu  kg  day , p < .0001 ) . paralleling these parameters , a statistically significant decrease in hba1c percentage ( 8.11      0.36 versus 6.16      0.21 , p < .0001 ) was found before and after islet transplantation ( figures 3(a ) , 3(b ) , 3(c ) ) . figure 4 shows a summary of the - hba1c values comparing nondiabetic , diabetic , and islet - transplanted nhps . there is a statistically significant difference in hba1c levels between nondiabetic and diabetic nhps and between nondiabetic and islet - transplanted nhps ( p < .0001 ) , showing a trend of improvement of hba1c values that parallels the improvement of blood glucose levels , as expected . however , caution is needed in interpreting these results because of the short time between samples . glycated hemoglobin ( hb ) is produced when a carbohydrate , such as glucose , binds to an hb molecule , such as hba0 , and undergoes intermolecular transformation to form a stable glycated ketoamine product . hba1c is formed via a posttranslational nonenzymatic attachment of glucose to hemoglobin at a rate dependent on the ambient blood glucose during the lifespan of the red blood cells ( approximately 120 days in humans ) . glycated hb percentage represents therefore the integrated value for average blood glucose concentrations in the preceding 6 to 8 weeks . a number of different glycated hb forms have been identified , reflecting various attached sugar residues . these forms include hba1a ( fructose-1,6-diphosphate , or glucose-6-phosphate ) , hba1b ( ketamine - linked pyruvic acid ) , and hba1c ( glucose ) . this last form is the most commonly tested , constituting approximately 80% of hemoglobin a1 ( hba1 ) . this hemoglobin is a derivate of adult hemoglobin ( hba ) , with monosaccharide ( fructose or glucose ) attachments . in strict chemical terms , the molecular structure of hba1c is n-(1-deoxy)-fructosyl - hemoglobin or n-(1-deoxyfroctose-1-yl ) hemoglobin beta chain . methods routinely used for the measurement of glycated hb percentage separate the molecule on the basis of its charge , structure , or antigenic properties . the most popular methods rely on the increased negative charge found in the glycated hb molecule to distinguish it from its nonglycated form . these assays include electrophoresis and ion - exchange chromatography as by high - performance liquid chromatography ( hplc ) . alternatively , boronate affinity chromatography separates glycated hb on the basis of its structure rather than its charge . in this assay , separation is the result of carbohydrate moieties on the glycated hb molecule binding by condensation to the affinity reagent , di - hydroxyboronate . this method is specific for all glycated hbs , irrespective of molecular charge or the site of glycation on the hb molecule . in addition , it is also able to detect the glycated portion of hb in patients with hb variants such as hb s , c , or f. thus , the term total glycated hb has been used to describe this type of assay . finally , immunoassays , such as the method we have utilized in this study , have been developed for the measurement of hba1c . monoclonal or polyclonal antibodies generated against the 6- to 8-amino acid peptide of the glycated amino terminus of the -globin chain are here utilized . advantages to measure hba1c percentage with this technology involve the simplicity of the approach ; the need for one small ( only few microliters ) blood sample ; the fact that the assay is relatively unaffected by recent food intake , activity , illness , or stress ; it still reflects glycemic control during the preceding few weeks   allowing measurement of hba1c even when pathological hemoglobin variants or nhp hemoglobins are involved . unfortunately , there are also a lot of different variables associated with measurement of glycated hb percentage that could potentially modify those values ; for example , processes that decrease the mean lifespan of rbcs will reduce the availability of hb for glycation and , therefore , the percentage of glycated hb , irrespective of glucose concentrations . similarly , conditions that increase the lifespan of rbcs ( e.g. , iron deficiency ) will increase the amount of glycated hb . appropriate validation is therefore required for experimental animal models such as diabetic nhp recipients of pig islet xenotransplantations . reliable measurement of glycated hb percentages would be then useful in determining the efficacy of therapeutic treatments . glycated hb percentages have been measured , only to a limited degree , in non diabetic , borderline - diabetic , and confirmed - diabetic nhps [ 17 , 2426 ] . percentages of hba1 and hba1c in nondiabetic baboons ( papio anubis ) , measured by use of cation - exchange chromatography , are approximately half of those found in humans . it has been suggested that this may be explained on the basis of differences in the life - span of rbcs in humans ( 100 to 120 days ) compared to baboons ( 30 to 60 days ) . at least 2 studies [ 25 , 26 ] have documented an increase in hba1c percentages in borderline - diabetic or confirmed - diabetic nhps . in one group of celebes crested macaques ( macaca nigra ) , the hba1c value increased from 2.6% in nondiabetic macaques to 7.9% in diabetic macaques , as measured by use of electrophoresis . in addition macaques without overt hyperglycemia , but with impaired glucose clearance , impaired insulin secretion , and increased postprandial glucose concentrations , had a significant increase in hba1c content to 3.5% . reference ranges for glycated hb percentages have not been determined in many nhps , but values that increase by 10% were generally considered to be abnormal . although the number of animals that were evaluated in our study is small , also reflecting the complexity of the experimental model , we had the opportunity to use the same technology to measure variations in hba1c values within the same animals under different clinical metabolic conditions : prior to diabetes induction , as insulin - dependent diabetics , and after islet transplantation . the levels of hba1c obtained reflected different glucose metabolic conditions such as diabetes , and improved metabolic control following islet transplantation . these values coherently represent the expected trends relative to the experimental metabolic conditions and further validate the technical approach proposed . by using this simple method in broader scale studies and employing larger numbers of animals , a better correlation between hba1c levels and blood glucose levels in cynomolgus monkeys in this study we presented the hba1c values of nondiabetic and diabetic nhps ( macaca fascicularis ) , analyzed with an inhibition of latex agglutination assay ( hba1c - specific mouse monoclonal antibody adsorbent onto latex particles ) . these values are lower than the ones we find in humans without diabetes and very similar   to those in diabetic patients under insulin treatment , respectively . total hba1c percentage can be measured with a user friendly assay and provides useful information for control of diabetes mellitus in nphs , as is done in human patients , and it could be used during the standard management of glucose metabolism in those animals . our data provide evidence that hba1c can be used to monitor diabetes in streptozotocin - diabetic monkeys and to receive feedbacks on the functionality of xenogenic islet transplantion , particularly useful during long - term followups .
non - human primates ( nhps ) are a very valuable experimental model for diabetes research studies including experimental pancreatic islet transplantation . 
 in particular nhps are the recipients of choice to validate pigs as possible source of pancreatic islets . 
 the aim of this study was to quantify glycated hemoglobin percentage in nhps and to assess whether changes in values reflect the metabolic trends after diabetes induction and islet transplantation . 
 sera from 15 nhps were analyzed . 
 9 nhps were rendered diabetic with streptozotocin ( stz ) , and 3 of them received porcine islet transplants . 
 hemoglobin a1c 
 ( hba1c ) 
 percentage was measured with an assay based on a latex immunoagglutination inhibition methodology . whereas diabetes and its duration were associated with increasing hba1c levels , postislet transplantation blood glucose normalization was paralleled by a decrease in the hba1c percentage . 
 our data provide evidence that hba1c is a useful tool to monitor glucose metabolism in nhps .