Journal of Diabetes and Its Complications xxx (2015) xxx–xxx

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Journal of Diabetes and Its Complications

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Measurement of the incretin hormones: glucagon-like peptide-1 andglucose-dependent insulinotropic peptide

Rune Ehrenreich Kuhre 1, Nicolai Jacob Wewer Albrechtsen 1, Bolette Hartmann,Carolyn F. Deacon, Jens Juul Holst⁎Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, DK-2200 Copenhagen, DenmarkDepartment of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark

Declaration of interest: The authors declare no finainterest that may have influenced the preparation of thi⁎ Corresponding author at: Department of Biomedical

Blegdamsvej 3B, DK-2200 Copenhagen N, Denmark.E-mail address: jjholst@sund.ku.dk (J.J. Holst).

1 Contributed equally to preparation of this manuscri

http://dx.doi.org/10.1016/j.jdiacomp.2014.12.0061056-8727/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Kuhre, R.E., etinsulinotropic peptide, Journal of Diabetes a

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 November 2014Received in revised form 6 December 2014Accepted 9 December 2014Available online xxxx

Keywords:Incretin hormonesGlucagon-like peptide-1Glucose-dependent insulinotropic peptideELISARIA

The two incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide(GIP), are secreted from the gastrointestinal tract in response to meals and contribute to the regulation ofglucose homeostasis by increasing insulin secretion. Assessment of plasma concentrations of GLP-1 and GIP isoften an important endpoint in both clinical and preclinical studies and, therefore, accurate measurement ofthese hormones is important. Here, we provide an overview of current approaches for themeasurement of theincretin hormones, with particular focus on immunological methods.

ncial or otherwise conflicts ofs review.Sciences, Panum Institute,

pt.

al., Measurement of the incretin hormonesnd Its Complications (2015), http://dx.doi.org

© 2014 Elsevier Inc. All rights reserved.

1. The incretin hormones and type-2-diabetes

The gut hormones, glucose-dependent insulinotropic polypeptide(GIP) and glucagon-like peptide-1 (GLP-1), are secreted in responseto nutrient intake and play an essential role for postprandial glucoseregulation by potentiating glucose-stimulated insulin secretion, aphenomenon called the incretin effect (Nauck, M.A. et al., 1986). Inhealthy humans, the effects of GIP and GLP-1 account for 25–70% ofthe total postprandial insulin response (depending on the size of theglucose load) (Creutzfeldt, 1979; Elrick et al., 1964; Perley & Kipnis,1967), but in type-2-diabetes, the incretin effect is greatly reduced orlost (Nauck, M., 1986). The loss is a specific and early characteristic oftype-2-diabetes development and is largely due to a reduction in theinsulinotropic effect of both hormones, although an impairment in thesecretion of GLP-1 may contribute (Holst et al., 2011; Nauck et al.,2011). Knowledge of the pathways involved in incretin hormonesecretion is therefore important for understanding the physiologicalmechanisms of blood glucose regulation in health and type-2-diabetes (Wewer Albrechtsen et al., 2014a). Accurate measurementof incretin hormones is not trivial, since plasma concentrations ofGLP-1 and GIP are low (in the low picomolar range). A low secretory

rate is responsible for this, but rapid post-secretory degradation of thepeptides and the existence of several peptide isoforms complicateanalyses further. In addition, there are inter-species differences withrespect to the molecular forms, requiring special attention, and finallythere is often unspecific interference by substances present inbiological fluids (Deacon & Holst, 2009). In the following, we willdiscuss current methods for quantification of GIP and GLP-1.

2. Methods for hormone quantification

Incretin hormones circulate in low (picomolar) concentrations,meaning that assays must have high sensitivity. This can be providedusing immune-based methods because it is possible to generateantibodies with equilibrium constants in the range of 1012 liters/mol,allowing reaction with femtomolar amounts of reagents (bioassaysmay have similar sensitivity, but often suffer from lack of specificity).Antibodies used in quantification assays are generally produced byimmunization of animals with fragments of the antigen coupled to alarger carrier protein in such a way that a certain region of the peptidecan be selected as immuno-determinant. This approach increases thelikelihood of directing antibodies towards a specific pre-definedepitope, and is useful for raising ‘region-specific’ antisera. Typically,the immunogen is administered together with an enhancer of theimmune response (e.g. Freund's adjuvant) on multiple (3–8)occasions separated by several weeks in order to boost the responseand increase the titer. Other methods may also be applied (e.g. phagedisplay technology) allowing for selection of not only regionspecificity, but also affinity (Winter et al., 1994). The phage display

: glucagon-like peptide-1 and glucose-dependent/10.1016/j.jdiacomp.2014.12.006

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technology is based on repeated rounds of antigen selection, phagepropagation (genetically engineered bacteriophages of antibody-library) and has particularly been applied in the development ofhuman monoclonal antibodies for therapeutic uses, e.g. anti-CD20 innon-Hodgkins Lymphoma (Hammers & Stanley, 2014).

The immune-based quantification of peptide hormones waspioneered in the 1950s by Berson and Yalow who described thefundamentals of radioimmunoassays (RIA) for quantification ofinsulin in humans (Berson et al., 1956). The principle is a competitionbetween radioactively labeled antigen (both free in solution) andunlabeled antigen (peptide standard or sample with unknownconcentration) for binding to a limited number of specific antibodybinding sites. Another technique, the ELISA technique (enzyme-linkedimmunosorbent assay) was developed more recently (Engvall &Perlmann, 1971) and has become the most frequently appliedprocedure for hormone quantification. ELISA is also based onantibody-binding, but unlike RIA, the read-out is based on anenzymatic reaction (e.g. horseradish peroxidase) coupled to one ofthe reagents and resulting in a colorimetric or otherwise traceablesignal (Engvall & Perlmann, 1971). The detection of the enzymaticconversion of a suitable substrate to a detectable metabolite is oftenbased on simple spectrophotometry (typically at 450 nm), but otherdetection systems (chemiluminescence, electrochemiluminescenceand fluorescence) exist. Compared to RIA, ELISA is generally morepracticable for the majority of laboratories for several reasons: (1)institutional regulations may discourage the use of radioactivity dueto safety concerns; (2) assays can be automated to allow high-throughput of samples because separation of free and bound ligands isobviated; (3) ELISA generally requires much lower sample volumesthan RIA, which also makes it suitable for small animal studies (anaveraged size mouse of 25 g contains only around 1 ml plasma); (4)the assay time of ELISAs is often less than that of RIAs and mostimportantly (5): with ELISA it is possible to target two epitopes of thesame peptide simultaneously by the so-called sandwich ELISAmethod. In sandwich ELISAs, two different antibodies are employed:one capture antibody typically pre-coated onto the assay plate, and adetection antibody coupled to one component of the enzyme-linkeddetection pathway. This improves specificity and accuracy; forexample, any truncated fragments arising from degradation will notbe detected if antisera directed against both the intact C- andN-termini of the molecule are used, making the assay specific forthe full-length hormone. This approach may also reduce non-specificinterference by plasma moieties, allowing quantification in untreatedplasma. In contrast, RIA and single-antibody ELISAs often requireplasma pretreatment, such as extraction by organic solvents (e.g.ethanol) or solid-phase (non-polar materials, C18) extraction toremove interfering substances (Bak et al., 2014a, 2014b). On the otherhand, the particular virtue of RIAs is that they are relatively simple andfast to develop, since they typically employ polyclonal antibodieswhich can be raised relatively easily. In contrast, ELISAs are generallybased on monoclonal antibodies, the raising of which is laborious andoften unsuccessful with respect to affinity (but, when successful, hasthe advantage of providing unlimited supplies of antibody).

More recently, novel approaches allowing for simultaneous quanti-ficationofmultiple analytes (termedmultiplexing)havebeen introduced.For example Luminex© has developed a technology based on antibody-coated magnetic beads (which are color coded), each of which binds toone of several (2–500) different hormones (depending on antibody) andis then detected by a specialized flow-cytometric technique. Although,clearly a brilliant concept, most multiplexing methods are challengedwith specificity and sensitivity issues (Bak et al., 2014a,2014b; WewerAlbrechtsen et al., 2014b), reducing their applicability, particularly withrespect to gut hormones. In the peptide hormone field, another methodunder development for hormone quantification is mass-spectrometry. Incontrast to the othermethodsmentioned,mass-spectrometry is based onidentificationof peptides according to theirmass, and should, inprinciple,

Please cite this article as: Kuhre, R.E., et al., Measurement of the ininsulinotropic peptide, Journal of Diabetes and Its Complications (2015),

be more specific as cross reactivity is not a concern. Currently, theapplicabilityofmass-spectrometry is limiteddue to insufficient sensitivity(usually around 100 pmol/l). However, the field is advancing rapidly, andover the past years, improvements in resolution/sensitivity (Michalski etal., 2012), sample preparation (Kulak et al., 2014), and computationalidentification of peptides (MaxQuant (Cox & Mann, 2008)) have beenmade, suggesting that mass-spectrometry based hormone quantificationmay not be far away.

3. Measurement of the incretin hormones

3.1. Measurement of GLP-1

GLP-1 is a highly conserved peptide hormone (100% homology acrossspecies) encoded by the proglucagon gene (GCG), expressed by L-cells inthe gut epithelium, certain neurons in the brain stem and by thepancreatic α-cell (Gu et al., 2013; Holst, 2007). The primary translationproduct of GCG is proglucagon, consisting of 160 amino acids, which iscleaved differentially according to tissue-specific expression of prohor-mone convertases (PC) 1/3 or PC2. Thus, in the L-cells, PC1/3 activityyields GLP-1, GLP-2, glicentin-related pancreatic polypeptide (GRPP),glicentin, and oxyntomodulin, while in the α-cells, the resultantfragments are GRPP, glucagon and the major proglucagon fragment(MPGF)as summarized inFig. 1 (Holst, 2007;Holst et al., 1994).However,circulating proglucagon products may also include PG 1–61 (“largeglucagon”), which may accumulate in renal disease (Baldissera & Holst,1986; Holst et al., 1983; Wewer Albrechtsen et al., 2014b), and theN-terminally extended forms of GLP-1, so-called GLP-1 1-36NH2/37(Orskov et al., 1994). Because of this complicated processing pattern andbecauseof the sequencehomologybetween theproducts, cross-reactivityis a significant challenge thatmust be dealtwithwhenmeasuring plasmaconcentrations of GLP-1 by immunological approaches. In addition, asalready mentioned, measurement of GLP-1 is complicated by lowconcentrations. In peripheral venous blood from fasted healthy humans,plasma concentrations of total GLP-1 (intact peptide + its primarymetabolite) are normally below 20 pmol/l, whereas the concentrationof the intact/biologically active formof GLP-1 accounts for amere fraction(usually 10–20%) of the total concentrations. This is due to the rapiddegradation (T1/2 ~ 1–2 min (Deacon et al., 1996; Vilsboll et al., 2003))by an enzyme, dipeptidyl-peptidase 4 (DPP-4), which generates theN-terminally truncated metabolite, GLP-1 9-36NH2/37.

3.2. Total GLP-1

When deciding which isoform(s) to measure, the choice shouldultimately depend on the research question investigated. If anestimate of L-cell secretion in response to a given stimulus/inhibitoris wanted, the best strategy for measurement is to employ an assaytargeting total GLP-1 (measuring both intact GLP-1 and the primaryGLP-1 metabolite). Since the DPP-4 enzyme cleaves the molecule atthe N-terminus, immunological methods for such measurementseither have to target epitopes in the mid-region of GLP-1 (so-called“side-viewing” antibody) or in the extreme C-terminal (“around thecorner”). As depicted in Fig. 1, “side-viewing” antibodies have anintrinsic problem of specificity, since the mid-region of GLP-1 is alsofound in MPGF from the α-cell and in GLP-1 1-36NH2/37. In contrastC-terminally targeting antibodies (“around the corner”) do notcross-react with MPGF, but this strategy raises another problembecause the C-terminal modification of GLP-1 varies among species.Thus, GLP-1 can undergo enzymatic processing (amidation) catalyzedby the peptidylglycine monooxygenase enzyme (PAM), wherebynon-processed (glycine-extended) GLP-1 7/9-37 is converted toGLP-1 7/9-36NH2. In humans and mice, the amide (NH2) isoform ofGLP-1 predominates (Orskov et al., 1994), but rats, pigs and the GLP-1producing cell lines GLUTag, NCI-H716 and STC-1 are all ‘partialamidators’, with just 2–5 times higher tissue concentrations of

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Fig. 1. Tissue specific processing of proglucagon. In the L-cells, proglucagon is processed by PC1/3 into GLP-1 (7-36NH2/7-37) GLP-2 and glicentin. By further PC1/3 activity, glicentincan then be further cleaved into glicentin-related pancreatic polypeptide (GRPP) and oxyntomodulin. In the pancreatic α-cell, proglucagon is processed by PC2 activity into GRPP,glucagon, major proglucagon fragment (MPGF) and to some extent to GLP-1 (1-36NH2/1-37). The potential processing products identified by antibodies raised against differentepitopes are indicated.

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amidated than non-amidated GLP-1 (Kuhre et al., 2014). Thus, whilereliable immunologically-based quantification of total GLP-1 secretionfrom humans and mice can be obtained by targeting amidated GLP-1,the determination of total GLP-1 secretion from pigs, rats and theGLP-1 producing cell lines GLUTag, NCI-H716 and STC-1 is moredifficult, because this requires either assays reacting with both of themolecular isoforms or the use of two different assays, one targetingthe amidated form and the other the glycine-extended molecularform. The use of two assays may be preferable, as experience from theauthors' laboratory suggests that commercial assays targeting bothforms may have differential recoveries of the amidated andglycine-extended forms and tend to overestimate concentrations ofintact GLP-1 due to cross-reactivity with the metabolite (9-36NH2/37(Bak et al., 2014b). Moreover, even within the same species, thedegree of GLP-1 amidation may vary, since the PAM enzyme is knownto be influenced by levels of copper and ascorbic acid (Bousquet-Mooreet al., 2009). The relevance of this for GLP-1 amidation is unclear, but,theoretically at least, variations in these nutritional factors could influencemeasurementsmade using C-terminally directed assays. The dynamics ofGLP-1 secretion from “partial amidators”may, in principle, be obtained bytargeting either of the molecular forms (amidated or non-amidated), butthe absolute total secretion of GLP-1 will be under-estimated.

3.3. Intact GLP-1

When is it relevant to measure the concentrations of intact GLP-1?Traditionally, measurement of the biologically active form wouldalways appear to be of interest, but not in the case of GLP-1. Studies ofthe release of GLP-1 from the porcine gut have demonstrated that thehormone is degraded locally by DPP-4 located on the endothelialmembranes of the capillaries draining the gut mucosa (Hansen et al.,1999). Up to 75% of GLP-1 may be degraded in this way even before itleaves the gut. Further degradation may take place in the liver(another 50% (Deacon et al., 1996)) and in the circulation, so that onlyabout 10% of what was originally released reaches the circulation(Hjollund, Deacon, & Holst, 2011). Immediately, it would seeminexpedient for the body to destroy a biologically active peptide sorapidly, but it turns out that GLP-1may interact with sensory afferentsof the parasympathetic nerve supply to the GI tract before it enters thecapillaries (Larsen & Holst, 2005). These neurons then transmit thesignals to the brain stem, where neurons of the nucleus of the solitarytract may transmit the signals further to the hypothalamus and to

Please cite this article as: Kuhre, R.E., et al., Measurement of the ininsulinotropic peptide, Journal of Diabetes and Its Complications (2015),

efferent pathways of the autonomic nervous system (Larsen & Holst,2005). This pathway, rather than the endocrine route whereby intactGLP-1 travels via the circulation, may be the predominant mechanismfor GLP-1 to exert its effects. Thus, the GLP-1 which is measured in thecirculation as the metabolite (GLP-1 9-36NH2/37) has probablyalready exerted its action via the nervous system, meaning thatmeasurement of total GLP-1 not only provides information about thesecretion of GLP-1, but also about its probable biological effects.Accordingly, measurement of peripheral circulating levels of intactGLP-1 may be less relevant, since this will provide information onlyabout the small fraction of the hormone that reaches its targets via theclassical endocrine route. Furthermore, this fraction is so small that itoften escapes detection (particularly after small stimuli), giving theimpression that the GLP-1 system was not activated at all. Neverthe-less, with large meals, there may be a measurable increase in theperipheral plasma concentration of intact GLP-1 (Vilsboll et al., 2003),and this increase may clearly influence the secretion of insulin fromthe islets after activation of the GLP-1 receptors on the β-cells.

However, there may be other experimental designs where itclearly will be relevant to measure the plasma concentrations of intactGLP-1. For instance, in studies investigating the effect of differentDPP-4-inhibitors, increases in intact GLP-1 levels will reflect theextent of DPP-4 inhibition; but if the investigator wants to examinethe degree of protection of GLP-1 fromDPP-4mediated degradation, itwill be necessary to measure also concentrations of total GLP-1. In thecase where intact GLP-1 is to be measured, the addition of a DPP-4inhibitor to the collection tubes is required to prevent furtherdegradation of the hormones ex vivo. Measurement of intact GLP-1requires an N-terminal antibody with an absolute requirement for thefree, unmodified N-terminus of the molecule and no cross-reactivitywith the primarymetabolites (9-36NH2/37) or N-terminally extendedforms. Here, a sandwich ELISA employing both N-terminal andside-viewing antibodies might be ideal, as it would at the same timeeliminate cross-reactivity with N-terminally truncated or elongatedforms, as well as being independent of the degree of C-terminalamidation (Deacon & Ahren, 2011). However, ideally, the side-viewing antibody should be directed towards the amino acids closestto the C-terminus of the molecule in order to avoid cross-reactivitywith any C-terminally truncated forms. Such forms may occur,because GLP-1 is a substrate for neutral endopeptidase (NEP) 24.11(Hupe-Sodmann et al., 1995), and increases in plasma levels of GLP-1can be measured with N-terminal specific assays after combined

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DPP-4 and NEP 24.11 inhibition (Plamboeck et al., 2005). We haverecently examined a wide range of commercially available immuno-based kits (RIA and ELISA) for quantification of intact and total GLP-1.We found pronounced differences in assay performance (sensitivity,specificity and precision) which emphasizes that the characteristics ofthe assay used should be taken into consideration when interpretingresults (Bak et al., 2014b).

3.4. Measurement of GIP

GIP is produced by the intestinal K-cells, which mostly reside inthe gut epithelium of the proximal small intestine, although they canbe found throughout the small intestine (Buchan et al., 1978;Mortensen et al., 2003). The GIP sequence is fairly well conservedacross species, withmore than 90% amino acid homology between thehuman, mouse, rat and porcine peptides. GIP is derived from a 153amino acid pre-pro-GIP precursor (Fig. 2) by PC1/3-dependentposttranslational processing (Ugleholdt et al., 2006), yielding bioac-tive 1–42 GIP (pre-pro-GIP 52–93) and flanking N- and C-terminalfragments, pre-pro-GIP 22–50 and 95–153, respectively, neither ofwhich is known to have any biological activity. Once released,bioactive GIP is degraded by DPP-4, producing the inactive, N-terminally truncated metabolite 3–42 (pre-pro-GIP 52–93) (Deaconet al., 2000). Although this degradation occurs at a slower rate thanthat of GLP-1 (resulting in a T1/2 of ~ 2 min in rodents and ~ 5–7 minin humans (Deacon et al., 2000; Kieffer, McIntosh, & Pederson, 1995),GIP 3–42 is still the major circulating form of GIP, accounting for up to60% of the total GIP immunoreactivity in human plasma (fasting levelapproximately 10–15 pmol/l) (Deacon et al., 2000). However, incomparison with GLP-1, the measurement of GIP is less problematic,since the processing pattern is less complicated (no amidation, noisoforms) and the circulating concentrations after stimulation arehigher. Commercial kits with ability to quantify both intact and totalGIP are available and, therefore, the same considerations apply as forGLP-1 when deciding which molecular form to measure; if anestimate of total secretion is wanted the best strategy is to measureGIP total (the sum of GIP 1–42 and GIP 3–42). In principle, suchmeasurements can be obtained by applying a ‘side-viewing’ antibodyor a C-terminally targeting antibody, both of which should be suitablesince no other products of the pro-GIP sequence that contain the sameimmuno-determinants (Fig. 2) seem to exist. Nevertheless, anantibody raised against GIP 1–42 (side-viewing) may not be idealfor analyzing GIP from different species because of amino acidvariations, and because it may cross-react with another largermolecule present in extracts of the small intestine; this moleculehas been designated 8-kDA GIP or GIP8000, although its structure

Fig. 2. Tissue specific pre-pro-GIP processing. In the K-cells, pre-pro-GIP is cleaved by PC1/3C-terminal domain (95–153). Cleavage of the signal peptide domain (1–21 pre-pro-GIP)theoretical processing products, that antibodies raised against different epitopes would tar

Please cite this article as: Kuhre, R.E., et al., Measurement of the ininsulinotropic peptide, Journal of Diabetes and Its Complications (2015),

remains unknown and it appears to have no known physiologicaleffects (Krarup, 1988; Krarup & Holst, 1984). Highly specificC-terminal directed antisera do not, however, cross-react withGIP8000 (Ugleholdt et al., 2006), and C-terminal targeting assaysare therefore most suitable for the measurement of total GIP. UnlikeGLP-1, GIP is not subjected toposttranslational amidation, eliminating thisproblemwhen using a C-terminal assay. For bioactive GIPmeasurements,an N-terminal directed assay that does not cross-react with GIP (3–42) isrequired and, as for GLP-1, a sandwich ELISA that employs an N-terminaltogether with a side-viewing or C-terminal targeting antibody mayincrease the specificity further. N-terminal assays appear to cross-reactwith another uncharacterized molecule, present in the fasting plasmawhich does not increase in response to K-cell stimulation (Deacon et al.,2000).Asa result, fastingconcentrationsmeasuredwithanassay for intactGIP may be higher than those measured with a C-terminal assay. Finally,unlike GLP-1, GIP is thought to act exclusively via the classical endocrinepathway, providing an argument to prefer assays for the intact peptide(the metabolite is inactive), if the aim is to address the impact of GIPsecretion for instance on β-cell secretion.

4. Discussion

Reliable measurement of incretin hormones is not an easy task andshould give rise to both theoretical and technical considerations.Common to all assays, the reliability of the measurements depends on4 basic criteria, namely sensitivity, precision, specificity and accuracy(the Richterich criteria) (Bak et al., 2014b; Wewer Albrechtsen et al.,2014b). As already discussed, sensitivity may present a definiteproblem since circulating concentrations may be very low, particu-larly of intact GLP-1, where levels are often below the lower limit ofquantification of the available assays. Precision may also present aproblem. For the immunoassays, intra-assay precision is usuallyaround 5% which may be tolerable, whereas inter-assay variation isoften considerable (15–20%). This means that comparisons of resultsshould only be carried out if the samples are analysed in the sameassay run. With only 70–80 wells usually available for unknowns/samples on an ELISA plate (35–40 if performed in duplicates), this is aserious problem, which is not easily dealt with. The accuracy is verydifficult to assess; in principle this should be evaluated by comparisonwith true values, but these are most often not known for endogenoushormones. Adequate accuracy implies satisfactory recovery of addedexogenous hormone, which is a must. Linear dilutions of samples withhigh concentrations are also required, and mixing of high and lowconcentration samples should give results that reflect the contributions ofthe samples. Validation by alternative physical–chemical methods, e.g.,chromatography is cumbersome and labor-intensive but may be

into GIP (1–42) and the flanking domains, i.e. the N-terminal pro-peptide (22–50) andoccurs independent of the PCs before pre-pro-GIP reaches the secretory vesicles. Theget (measure), are indicated.

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rewarding. As briefly mentioned application of mass spectrometry mayturn out to be very helpful. Unfortunately, very few commercial assayshave been subjected to thorough reliability analysis.

For immuno-based methods, the inter-assay variation of mostcommercial assays is typically 10% or more. In larger studies, wherethe entire sample collection cannot be measured on a single ELISAplate or in a single RIA, it is necessary to include quality controls ineach assay run (e.g. a plasma sample with a known concentration ofthe analyte) for determination of the inter-assay variation. Commer-cial assays often indicate an “acceptable” range of results for qualitycontrols, but this range is usually very wide. Should one adjust themeasured values according to the result of the quality controlmeasurement? This is generally not recommended. It may be usefulto employ internal standards for each individual/patient (i. e. additionof a known amount of peptide to a fasting plasma sample from thatindividual) which could be used for adjustment for inter-assayvariation. In order to minimize the influence of inter-assay variation,all samples from larger studies should be measured using assays withsame batch number. In addition, all samples from the same patientshould be measured in the same assay run. However, the benefits ofavoiding or minimizing high inter-assay variation must be heldagainst the potential deterioration of GLP-1 and GIP upon long-termstorage (for instance, obtaining samples from before and after surgicalor other intervention inevitably results in prolonged sample storagebefore analysis). As the long term-stability of GLP-1 or GIP has not hasbeen investigated adequately, this may be a significant obstacle that isdifficult to circumvent. Observations from the authors' laboratory do notindicate that significant loss of immunoreactive GLP-1 occurs duringstorage of humanplasma samples (containing aDPP-4 inhibitor) for up to1 year (at −20 °C or −80 °C). In contrast, handling conditions prior tothe actual assay did affect the measured concentrations of intact GLP-1,but not total GLP-1, meaning that storage on ice during the analyses andthe addition of a DPP-4 inhibitor to the analytical tubes to preventdegradation of intact GLP-1 are absolutely required. At present, no suchdata are available for GIP, although it is the experience of the authors thatGIP levels change little with time, and that conditions for samplepreparation may not be critical since similar values were obtained inplasma and serum samples.

The limited sample volume that can be obtained in rodent studieshas long been an analytical challenge, but the development andimprovement of ELISA based methods and multiplexing technology

Table 1Ten considerations when measuring incretin hormones.

How best to approach your research question; are you interested in L/K-cell secretion,then use assays for “total” concentrations. Only measure the intact isoforms if the intactisoforms are of specific interest.Have the samples been pretreated optimally: with DPP-4 inhibitor(for intact assays) and on ice (all assays)?

Preferably measure all your samples at the same time and if possible insame assay run (reduces inter-assay variation).

Use assay quality controls for each run—if not provided by themanufacturer, prepare plasma pool with known amounts of peptideand include an aliquot in each assay run.

Test the kit before use: does the assay perform well in your samplematrix (plasma, serum, lymph fluid, cerebrospinal fluid, urine,cell culture medium, etc.)?

Which detection methods can be used at your department? Do you haveaccess to a gamma-counter, spectrophotometer, etc.?

How much sample volume do you have, and do you plan to have enoughplasma for duplicate determination (reduction of intra-assay variation)?

Do the samples need to be diluted to stay within the working range of the assay(for instance during infusion studies)? If so, can samples at different dilutionsbe compared? (are the dilutions linear?)

Has the assay been sufficiently validated? And is detailed documentationof this available?

Does the department have any former expertise in measurement—considerseeking external advice or collaboration to ensure reliableassessment of concentration.

Please cite this article as: Kuhre, R.E., et al., Measurement of the ininsulinotropic peptide, Journal of Diabetes and Its Complications (2015),

shows promise as they require much smaller sample volumes thanRIAs (down to 1 μl). However, currently these assays seem to lacksufficient sensitivity for detecting small changes in concentrations ofintact GLP-1 or GIP (Bak et al., 2014a).

Therefore, in the planning of an experiment that includesmeasurements of the incretin hormones, a number of issues shouldbe taken into consideration to optimize the validity of the results. Themost important of these are summarized in Table 1. In the future,other approaches, such as mass-spectrometry based proteomics, mayhelp by eliminating the immuno-specificity issues, but currently, theirlack of sensitivity prevents their general use for analysis of plasmasamples. Therefore, for the time being, RIA and ELISA remain the mostefficient and powerful methods for hormone quantification.

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