The kidneys play an important role in homeostasis, they regulate the amount of water and salts present in the body by filtering blood through the nephrons. Waste products are filtered out and eliminated from the body in the urine, which is made up of the excess water, salts and waste products.
When the kidneys are not functioning efficiently, waste products and fluids begin to accumulate instead of being excreted which can cause serious health problems. Furthermore, kidney disorders can often develop and advance over a period of time without showing any signs; alternatively, symptoms are not recognized as being associated with kidney problems. Kidney function testing is therefore relevant for diagnosing and monitoring disease and assists in the development of appropriate treatment plans. Laboratory automation facilitates the efficiency and productivity of clinical laboratories. The determination of parameters related to kidney function by using tests incorporating reagents applicable to a variety of automated analysers facilitates clinical effectiveness and patient outcomes when managed by qualified laboratory professionals.
Kidney function assessment
Many conditions can affect the ability of the kidneys to carry out their vital functions. Some conditions can lead to a rapid (acute) decline in kidney function; other conditions lead to a gradual (chronic) decline. A number of clinical laboratory tests in blood and urine can be used to assess renal function. The unit measure of kidney function is the glomerular filtration rate (GFR), which can be defined as the volume of plasma cleared of an ideal substance –freely filtered at the glomerulus and neither secreted nor reabsorbed by the renal tubules- per unit of time. The normal range is 80-120 ml/min. Measuring this rate is a laborious process. Creatinine is the closest to an ideal endogenous substance for measuring GFR.[1,2] Creatinine is derived from creatine and creatine phosphate in muscle tissue and is defined as a nitrogenous waste product. Creatinine is not reutilized but is excreted from the body in the urine via the kidney. As a consequence of the way in which creatinine is excreted by the kidney, its measurement is used almost exclusively in the assessment of kidney function.
Urea, a byproduct of protein metabolism, is produced in the liver and then is filtered from the blood and excreted in the urine by the kidneys. The blood urea nitrogen test (BUN) measures the amount of nitrogen contained in the urea, high levels can indicate kidney dysfunction. As these levels are also affected by protein intake and liver function, this test is usually done together with a blood creatinine test.
Cystatin C is a small cysteine proteinase inhibitor that is steadily produced by all nucleated cells. The small molecular weight of cystatin C allows it to be freely filtered by the glomerular membrane and therefore cystatin C levels in the blood are indicative of a normal or impaired GFR. Levels of cystatin C in serum/plasma are almost entirely dependent on GFR.[3]
Other tests for the measurement of other parameters regulated in part by the kidneys can also be useful for the evaluation of kidney function; these tests include electrolytes (sodium, potassium, chloride, bicarbonate), protein, uric acid and glucose:
Application of kidney function tests to automated systems
In clinical settings the application of tests for the determination of parameters related to kidney function to automated systems, facilitates clinical effectiveness and productivity. There are currently tests available for the determination of creatinine, BUN, cystatin C, electrolytes, protein, uric acid and glucose among others. If a variety of these tests could be applied to one system, the result output for each system would increase, which would maximize efficiency. The use of tests incorporating reagents applicable to a variety of automated analysers is beneficial as it increases the testing capacity of one system. This is further enhanced by the analyser’s capability to employ different methodologies with different reagents. The combination of automation and the use of stable, high performance reagents, lead to optimal analytical performance, extensive measuring ranges to ensure detection of abnormal values and reduced interference to produce more accurate results. For instance, a study using a creatinine test reported no interference with bilirubin and metamizol.[6]
The application of other kidney function related tests to studies in patients with nephrotic syndrome, chronic liver diseases and diabetes have also been reported.[7-8]
The automation of laboratory testing still requires qualified laboratory professionals for the evaluation of the results but reduces errors, staffing concerns and safety issues. This facilitates the diagnosis and the monitoring of kidney function, which is of great importance in clinical practice and in research.
Conclusion
The kidneys are the body’s natural filtration system and perform many vital functions. Kidney function tests is a collective term for a variety of individual tests and procedures for the evaluation of how well kidneys are functioning. The determination of parameters related to kidney function (i.e.creatinine, BUN, cystatin C, electrolytes, protein, uric acid, glucose) by using tests incorporating reagents applicable to a variety of automated analysers, increases the testing capacity of the systems and facilitates clinical effectiveness and patient outcomes when managed by qualified laboratory professionals.
References
1. Berger A. Renal function – and how to assess it. BMJ. 2000; 321: 1444.
2. Traynor J, Mactier R, Geddes CC, Fox JG. How to measure renal function in clinical practice. BMJ. 2006; 333 (7571): 733-737.
3. Laterza OF, Price CP, Scott MG. Cystatin C: an improved estimator of glomerular filtration rate? Clin. Chem. 2002; 48(5): 699-707.
4. Kirby M. Screening for microalbuminuria. The British Journal of Diabetes and Vascular Disease. 2002; 2(2): 106-109.
5. Sechi LA, Catena C, Zingaro L., Melis A, De Marchi S. Abnormalities of glucose metabolism in patients with early renal failure. Diabetes. 2002; 51: 1226-1232.
6. Harmonien AP. Bilirubin and metamizol do not interfere with the Randox enzymatic creatinine test. An evaluation of a new enzymatic creatinine determination method. Eur. J. Clin. Chem. Clin. Biochem. 1996; 34(12): 975-976.
7. Mula-Abed W-AS and Hanna BE. Measurement of serum fructosamine as an index of glycated protein in patients with nephrotic syndrome and chronic liver diseases. Bahrain Medical Bulletin 2001; 23(4).
8. Hirnerova E, Krahulec B, Strbova L, Stecova A, Dekret J, Hajovska A, Ch A Dukat A. Effect of vitamin E supplementation on microalbuminuria, lipid peroxidation and blood prostaglandins in diabetic patients. Bratisl. Lek. Listy 2004; 105(12): 408-413.
Author
María Luz Rodríguez
Randox Laboratories Limited,
55 Diamond Road, Crumlin,
County Antrim, N. Ireland, BT29 4QY,
United Kingdom
Therapeutic drug monitoring of anti-epileptic drugs has greatly advanced since the development of colorimetric assays for the measurement of phenytoin and phenobarbital in the mid-1950s. Today, not only have laboratory technology and assay development advanced, but so have the pharmaceutical agents available for the treatment of epilepsy disorders. However, under UK National Institute for Health and Clinical Excellence (NICE) Guidelines, therapeutic drug monitoring is still justified for newer anti-epileptic drugs like levetiracetam and pregabalin, for which we have developed quick and robust LC-MS/MS assays.
by Jonathan C. Clayton, Katherine Birch and Carrie A. Chadwick
Background
Therapeutic drug monitoring (TDM) is an important consideration in the treatment of epilepsy. It has long been known that a dose of a given drug may be effective in one patient but not in another [1]. This is of particular importance when too high a concentration of drug can have toxic effects, and too low a concentration has no therapeutic effect. Problems arise when, in different patients, a specific dosage leads to a therapeutically significant concentration in one, but could be ineffective or even toxic in another. Understanding the relationship between dosage and the concentration of the active drug at receptor sites has long been a topic for research [2], which has led to the development of assays to measure the plasma concentration of anti-epileptic drugs (AEDs). TDM of AEDs has advanced since colorimetric assays for phenytoin and phenobarbital were developed in the mid-1950s [3]. Older AEDs such as phenytoin and valproate have narrow therapeutic ranges (the plasma drug concentration range below which the drug may be ineffective and above which the patient may experience toxic effects). However, even the plasma concentration at which a given drug is effective may vary from individual to individual, depending on a number of factors known as pharmacokinetics [4]. Many newer AEDs, such as lamotrigine and topiramate do not have the narrow therapeutic range as seen with the older AEDs, however, TDM is still applicable [5]. Today both older AEDs such as phenytoin, phenobarbital and sodium valproate as well as newer AEDs such as lamotrigine and topiramate are subject to TDM [4]. This has led to the development of new assays for monitoring the serum concentration of these drugs. Methods include immunoassays such as enzyme multiplied immunoassay technique (EMIT) and cloned enzyme donor immunoassay (CEDIA), kinetic interaction of microparticles (KIMS) and chemiluminescent assays (CLIA) [6]. However, more liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are being developed for newer AEDs, which can detect a number of AEDs in a single assay [7].
Best Practice Guidelines for TDM published in 2008 [1], along with a review discussing TDM of the newer AEDs [8] have provided a rationale for developing methods for two second generation AEDs, levetiracetam and pregabalin. These drugs are becoming increasingly popular with levetiracetam being used as an adjunct for partial and generalized tonic–clonic seizures, and pregabalin used as an adjunct for partial seizures [9]. Pregabalin, and to a lesser extent levetiracetam, is also used in the treatment of non-epileptic disorders such as neuropathic pain [9]. The increasing popularity of these drugs with clinicians has led to an increasing demand for determination of plasma concentrations of these drugs. TDM is justified for determining compliance with treatment with either drug, but also for determining overdosing, and dosing in renal failure, of levetiracetam.
Here, we describe methods for the detection and quantification of levetiracetam or pregabalin in serum using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The methodology is identical for both levetiracetam and pregabalin and so, should demand for TDM of these drugs increase in the future, there is scope for them to be combined into one assay.
Materials and methods
Levetiracetam (1 mg/mL in MeOH) and pregabalin (1 mg/mL in MeOH) stock solutions, levetiracetam-D6 (100 µg/mL in MeOH) and pregabalin-D6 (100 µg/mL in MeOH) were purchased from Cerilliant (distributed by LGC Standards, Middlesex, UK). EQA materials used for accuracy assessment were kindly supplied by the LGC Heathcontrol EQA scheme. HPLC grade water and methanol were purchased from Sigma-Aldrich Ltd (Poole, Dorset, UK). All other chemicals were purchased from Sigma-Aldrich Ltd or VWR Ltd. ClinChek® Control Levels 1 and 2 were purchased from RECIPE (Munich, Germany).
Standards
Standard solutions were made by preparing serial dilutions of stock solution in PBS/BSA (phosphate buffered saline containing 0.5% bovine serum albumin) (137 mmol/L NaCl, 2.7 mmol/L KCl, 5.4 mmol/L Na2HPO4•7H2O, 1.8 mmol/L KH2PO4, 0.5% BSA). The standards were stored at –20°C until use.
Internal standards
Each internal standard was prepared to a final concentration of 10 mg/L in HPLC grade methanol containing 50 mmol/L ZnSO4∙7H2O. The internal standards were stored at room temperature until use.
Sample preparation
For assay purposes, standards, quality control (QC) and serum samples were prepared in an identical fashion. In a 96-well plate, 80 μL internal standard solution (in ZnSO4 in MeOH) are added to 20 μL sample followed by agitation and centrifugation. Eighty microlitres of H2O was then added to each well, the plate heat sealed, agitated and centrifuged.
Chromatography and mass spectrometry
Chromatography was performed on a Waters Acquity UPLC system equipped with a Waters Acquity UPLC BEH C18 1.7 μm 2.1 x 50 mm column. Mobile phase A consisted of 10 mmol/L ammonium acetate and mobile phase B consisted of MeOH.
A flow rate of 0.5 mL/min was maintained for the run time of 2.5 minutes. A linear gradient of mobile phase B from 2% to 50% was run between 0 and 1 minutes, followed by a constant concentration of 50% mobile phase B. Ninety-eight per cent mobile phase B was run from 1.75 to 2.5 minutes. The injection volume was 5 μL.
Mass spectrometric determination was carried out using a Waters TQD in ESI+ mode. The source temperature was 130 °C, desolvation temperature was 400 °C, cone gas flow was 50 L/hr and the desolvation gas flow was 800 L/hr. Targetlynx™ software was used to process the data and quantify the drugs in the standards, controls and patient samples.
Method validation
Validation of the assays was carried out according to Honour [10]. Precision and bias were determined by measuring QC samples over 5 batches with 5 samples in each batch. The coefficients of variance (CVs) were calculated for intra-batch and inter-batch precision. Bias was calculated from the nominal target values for each of the QC materials.
Accuracy was assessed using EQA materials from the LGC Heathcontrol AE1 Anti-epileptic drug EQA scheme.
Matrix effects were determined by running a water blank, extracted water and extracted drug-free serum against a background infusion of each drug.
The limit of blank (LOB) was determined by running 10 extracted water samples and was quantified as the highest concentration measured in the absence of analyte.
The lower limit of quantitation (LLOQ) was determined by spiking drug-free serum with known quantities of each drug, and was quantified as the lowest detectable concentration whose CV was <15% and bias <20%.
Specificity was determined by spiking PBS/BSA with high concentrations of six more commonly used AEDs (carbamazepine, carbamazepine epoxide, phenobarbital, phenytoin, primidone and sodium valproate.
Carry-over was determined by spiking drug-free serum with high concentrations of each drug, and analysing followed by drug-free serum.
Results
Chromatography and mass spectrometry
Levetiracetam and levetiracetam-D6 had a retention time of 0.88 minutes and the cycle time from injection to injection was 3 minutes. Pregabalin and pregabalin-D6 had a retention time of 0.82 minutes and the cycle time from injection to injection was 3 minutes. The chromatography profile is identical for both of the drugs. The profile produced clean, sharp peaks with no co-eluting elements. The quantification transition for levetiracetam was m/z 170.90>69.16 and the confirmation transition was m/z 170.90>98.17. For pregabalin, the quantification transition was m/z 159.90>55.12 and the confirmation transition was m/z 159.90>83.08. For the internal standards, levetiracetam-D6 had the transition m/z 177.00>132.00 and pregabalin-D6 had the transition m/z 166.10>102.90.
Method validation
The intra- and inter-assay CVs are <8% for both drugs suggesting good precision of the assay. The inter- and intra-assay bias for levetiracetam was acceptable at <6%, while for pregabalin the inter- and intra-assay bias was <10% apart from the inter-assay bias at 10 mg/L (Table 1). External quality assessment materials were analysed as per patient samples. The results (Table 2) were compared with the target value supplied by LGC Heathcontrol, and with the returns of other laboratories using similar methods (LC-MS and LC-MS/MS) in order to determine the accuracy of the assay. Matrix effects were investigated using injections of drug-free serum, extracted water and blank water against a constant background infusion of each drug in methanol (50 mg/L levetiracetam, 25 mg/L pregabalin). No matrix effects are seen around the relevant retention times for either drug (Fig. 1). The LOB was quantified as the highest apparent analyte concentration in the absence of analyte. The LLOQ was quantified as the lowest level of analyte detectable whose CV was <15% and whose bias was <20% (Table 3). The methods for both levetiracetam and pregabalin showed no interference from any other commonly prescribed AEDs, with responses of ‘0’ to the interference samples from both methods. Blank serum samples and extracted water samples run immediately after samples containing either ~200 mg/L levetiracetam or 100 mg/L pregabalin gave responses of ‘0’, indicating no problems with carry-over.
Discussion
We have developed and validated LC-MS/MS assays for the quantification of levetiracetam and pregabalin in serum.
Two optimal transitions were identified for both drugs, thus providing a ‘quantifier’ transition and a ‘confirmation’ transition in order to increase confidence of identification owing to the risk of misidentification of analytes with the same molecular weights as the drugs of interest.
The chromatography method is identical for both levetiracetam and pregabalin, and with the two drugs having different retention times (0.88 and 0.82 minutes respectively), should there ever be a wish to combine these assays into one single run, this should be straightforward. Additionally, should assays for any other AEDs be developed, this chromatography method would be an appropriate starting point. Serum proteins are precipitated by the addition of ZnSO4 in methanol, which also aids the retained solubility of the drug. Following centrifugation, an equal volume of H2O is added so the drug is in 50 : 50 methanol/water. Following a further centrifugation, 5 µl of supernatant is injected onto the column. The method is quick and robust. The assay has acceptable precision and bias. All the EQA materials ran well within their acceptable ranges, close to the target value.
Other LC-MS/MS methods for the detection of levetiracetam [11, 12] and pregabalin [13] have been described, all of which have longer cycle times between injections, larger sample volume requirements, and, in some cases, have more complex sample preparation. The method described here benefits from being quick, with a simple sample preparation procedure.
Methods for the measurement of levetiracetam in saliva have been described [11] and it has been shown that there is good correlation between saliva, plasma and serum, meaning saliva would be a suitable alternative to serum [14]. To date, no such method has been described for pregabalin, but cases of pregabalin toxicity have been described which would advocate the development of further methods for the TDM of pregabalin [14].
The monitoring of levetiracetam and pregabalin is justified [1, 5] to monitor compliance and overdosing, and quick and robust methods for their measurement in serum have been described here. Further work could include development of assays for the measurement of these drugs in saliva, with comparison studies required.
References
1. Patsalos PN, Berry DJ, Bourgeois BF, Cloyd JC, Glauser TA, et al. Antiepileptic drugs—best practice guidelines for therapeutic drug monitoring: a position paper by the subcommission of therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies. Epilepsia 2008; 49: 1239–1276.
2. Eadie MJ. Therapeutic drug monitoring—antiepileptic drugs. Br J Clin Pharmacol. 1998; 46: 185–193.
3. Theodore WH. Rational use of antiepileptic drug levels. Pharmac Ther. 1992; 54: 297–305.
4. Glauser TA, Pippenger CE. Controversies in blood-level monitoring: reexamining its role in the treatment of epilepsy. Epilepsia 2000; 41(Suppl. 8): S6–S15.
5. National Institute for Health and Clinical Excellence. The epilepsies: the diagnosis and management of the epilepsies in adults and children in primary and secondary care. Clinical guidelines 137. NICE 2012; http://guidance.nice.org.uk/CG137 (accessed 15 October 2013).
6. Aldaz A, Ferriols R, Aumente D, Calvo MV, Farre MR, et al. Pharmacokinetic monitoring of antiepileptic drugs. Farm Hosp. 2011; 35: 326–329.
7. Shibata M, Hashi S, Nakanishi H, Masuda S, Katsura T, Yano I. Detection of 22 antiepileptic drugs by ultra-performance liquid chromatography coupled with tandem mass spectrometry applicable to routine therapeutic drug monitoring. Biomed Chromatogr. 2012; 26: 1519–1528.
8. Krasowski MD. Therapeutic drug monitoring of the newer anti-epilepsy medications. Pharmaceuticals 2010; 3: 1909–1935.
9. Wahab, A. Difficulties in treatment and management of epilepsy and challenges in new drug development. Pharmaceuticals 2010; 3: 2090–2110.
10. Honour JW. Development and validation of a quantitative assay based on tandem mass spectrometry. Ann Clin Biochem. 2011; 48: 97–111.
11. Guo T, Oswald LM, Mendu DR, Soldin SJ. Determination of levetiracetam in human plasma/serum/saliva by liquid chromatography-electrospray tandem mass spectrometry. Clin Chim Acta 2007; 375: 115–118.
12. Blonk MI, van der Nagel BC, Smit LS, Mathot RA. Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2010; 878: 675–681.
13 Nirogi R, Kandikere V, Mudigonda K, Komarneni P, Aleti R. Liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry method for the quantification of pregabalin in human plasma. J Chromatogr B. 2009; 877: 3899–3906.
14. Patsalos PN, Berry DJ. Therapeutic drug monitoring of antiepileptic drugs by use of saliva. Ther Drug Monit. 2013; 35: 4–29.
The authors
Jonathan Clayton* MPhil, MSc; Katherine Birch DipRCPath; and Carrie Chadwick FRCPath
The Buxton Laboratories, The Walton Centre NHS Foundation Trust, Liverpool, UK
*Corresponding author
E-mail: Jonathan.clayton@nhs.net
Medical care has been undergoing tremendous advances in the Middle East, not least in diagnostic testing. Roche Diagnostics Middle East (RDME) is a pioneer in leading this development in in vitro diagnostics (IVD), by supporting laboratories to achieve a higher level of performance, efficiency and sustainability. IVD testing directs over 60% of clinical decision-making and accounts for a small fraction of global healthcare spending. Whether it is in oncology, virology, blood screening, or research on infectious diseases, autoimmunity, inflammation, women’s health or metabolism, RDME has supported a large number of leading and prominent healthcare institutions to move from multiple analyzers and workflows to comprehensive, integrated laboratory solutions, meeting international standards and certifications.
Roche, with its unique privilege of having both pharmaceutical and diagnostics research under one roof, aims to improve healthcare and make a difference in patients’ lives. The medical solutions start from the stages of early detection and prevention of disease, to diagnosis, treatment selection and treatment monitoring. Roche Diagnostics is leading the industry by addressing unmet medical needs with new or medically enhanced diagnostic tests, supporting doctors and patients with an improved information basis for better medical decisions and treatment selection. Roche’s IVD test menu is one of the broadest in the industry and is continually being expanded based on the latest scientific advances.
Pioneering in Personalized Healthcare
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Leading in optimizing performance
Optimizing performance, automation and information technology are simplified with a common architecture that delivers tailor-made solutions for diverse workloads and testing requirements. Roche Diagnostics offers platforms that are designed to reduce the complexity of laboratory operation and provide efficient and compatible solutions for network cooperation. For example, Roche Diagnostics has developed medical diagnostic tests based on the Nobel prize-winning polymerase chain reaction (PCR; which exponentially amplifies small amounts of target DNA), that would otherwise be too time-consuming or impossible to perform.
Providing superior workflow solutions, including blood screening
Superior workflow solutions such as Task Targeted Automation (TTA) and Total Lab Automation (TLA) are designed to meet the needs of today’s fast developing healthcare systems. In RDME, a regional project management team is an added value to customers by providing consultancy and implementation support. TLA is customized to the specific needs of individual customers and, thanks to the modular system landscape, can be configured in 90 layouts, differing in size and shape. Roche Diagnostics is successfully delivering best in class Total Lab Solutions for Pathology and Cytology Laboratories to substantially improve the workflow with a unique and complete solution. Another example of superior workflow solutions and automation is with blood screening. Roche Diagnostics has been the preferred partner in the Blood Bank Industry by safeguarding patients through industry-leading assays and technologies. Besides offering Nucleic Acid Testing (NAT), Roche Diagnostics launched Roche Blood Safety Solutions (RBSS), which introduced serology testing of blood samples in an automated manner. As such, Roche Diagnostics is the only provider of a complete Blood Safety Solution to blood banks of any size. Fully integrated automation is offered; these standardized processes reduce manual steps, which guarantees the safety of the blood supply and offers state-of-the-art assay sensitivity and genotype coverage.
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Similarly, Roche offers centralization of data which is achieved with rapid and easy-to-operate systems that facilitate immediate healthcare decisions, thus placing an emphasis on patient-oriented diagnosis. One example of such a system is the Cobas IT 1000, a point-of-care IT solution that provides complete remote management of and access to all point-of-care diagnostic systems from just one hospital workstation. This automation and centralization of data management into just one workstation frees staff time and enhances the diagnostic service offered to patients.
Roche Diagnostics’ ongoing commitment to developing new analytical tools greatly benefits patients, and its technological innovations create a big impact on the healthcare development in the Middle East. As well as the analytical and technological advances described above, RDME has worked hard to establish the relevant infrastructure in the Middle East with a logistics hub, continual training for employees and a customer support center. These factors combined make RDME the leader in IVD and allow healthcare professionals to benefit from reliable, accurate and immediate results, which directly impact their diagnoses. RDME provides the deepest industry know-how and aims to become the region’s trusted IVD partner.
Fibroblast growth factor-23, a key regulator of phosphate and 1,25-dihydroxyvitamin D metabolism, appears to be an independent risk factor for mortality among chronic kidney disease patients. However, sample stability and poor analytical agreement between detection methods still need to be addressed for it to become a reliable biomarker.
by Dr A. Kumar, Dr W. Herrington, Dr S. Clark and Dr M. Hill
The function of fibroblast growth factor-23 and its role in chronic kidney disease
Fibroblast growth factor-23 (FGF-23) was identified as the key regulator of phosphate homeostasis from a study of renal phosphate-wasting condition autosomal dominant hypophosphatemic rickets [1]. It is secreted principally by bone-forming osteocytes and osteoblasts in response to increased dietary phosphate intake and abnormally elevated serum phosphate concentration (hyperphosphatemia). FGF-23 acts to correct raised phosphate levels by increasing urinary phosphate excretion, by direct inhibition of renal tubular phosphate reabsorption, and via reducing dietary phosphate absorption, by suppressing 1α-hydroxylation of 25-dihydroxyvitamin D to form active 1,25-dihydroxyvitamin D [1,25(OH)2D]. Low 1,25(OH)2D production also provides a negative feedback signal in phosphate homeostasis by inhibiting further FGF-23 secretion (Fig.1)[2, 3].
Chronic kidney disease (CKD) commonly causes a fall in glomerular filtration rate resulting in a reduced capacity for phosphate excretion [4]. FGF-23 levels increase early in CKD, often before any detectable rise in phosphate concentration [5] and those with the severest form of CKD, end-stage renal disease (ESRD), have FGF-23 levels that are 100 to 10,000-fold higher than healthy controls [4, 6]. Sustained suppression of renal 1,25(OH)2D synthesis by high FGF-23 levels contributes to lower serum calcium concentration, a stimulant of parathyroid hormone (PTH) secretion. PTH maintains normal serum calcium concentration by promoting reabsorption of the calcium from its reservoir in bone. The abnormal elevated levels of FGF-23 seen in CKD thus results in a disruption of the homeostatic balance of calcium and phosphate and this may impact on many normal processes including bone mineral metabolism and cardiovascular function. The consequences of prolonged derangement of bone mineral metabolism (known as CKD-mineral bone disease; CKD-MBD) is bone pain and increased fracture risk. CKD-MBD may also accelerate calcification of the vascular tree, a process that may explain some of the significantly increased cardiovascular risk in those with CKD [7]. Indeed, among CKD patients, several studies have shown FGF-23 to be an independent risk factor for mortality [8] and among those not on dialysis it appears to also predict CKD progression [4, 9]. Treatments that might positively impact on FGF-23 levels, for example, reducing dietary phosphate absorption with phosphate binders may therefore have beneficial effects on bone, renal and cardiovascular outcomes in those with CKD (both in those with hyperphosphatemia and those with high-normal serum phosphate concentration).
Methods for FGF-23 assessment
In vitro studies have shown that some of the FGF-23 synthesized by the osteocytes is cleaved between amino acid 179 and 180 by furin (a type I precursor convertase) releasing a C terminal fragment. Current immunometric methods detect either ‘intact’ FGF-23 (iFGF-23, ~32 KDa) or ‘C-terminal’ fragments (cFGF-23, ~14 KDa) in plasma or serum. The cFGF-23 assays recognize two epitopes in the C-terminus, thereby recognizing both iFGF-23 and cFGF-23 fragments. The intact assays recognize only the iFGF-23 because the epitopes flank the cleavage site [2]. At present, there is no reference method, or consensus to indicate which assay type is the most suitable for measuring circulating FGF-23. If all circulating FGF-23 is intact and biologically stable, concentrations detected by the intact and C-terminal assays should be comparable [10]. However, there is a paucity of data confirming this and so caution should be used when comparing studies using the different methods.
Several studies have assessed the performance of commercially available enzyme-linked immunosorbent assays (ELISAs) for FGF-23. Heijboer and co-workers evaluated the performance of one cFGF-23 assay (Immutopics, USA) and two iFGF-23 assays (Immutopics and Kainos Laboratories Inc., Japan) using samples from healthy volunteers and patients with expected high levels of FGF-23 [11]. Intra- and inter-assay variations were assessed in approximately 100 samples with low, normal and high FGF-23 concentrations providing <20% CV for the cFGF-23 Immutopics and iFGF-23 Kainos assays. A high intra-assay variation (22–61%) was observed for the Immutopic intact assay which may be due to lot-to-lot variation [11]. A potential difficulty observed with the Kainos intact assay is poor assay performance when using an automated plate washer, as directed in their protocol. Heijboer and co-workers found acceptable results were only obtained when the wells were washed manually, which made this method impractical for measurement of large numbers of samples [11]. However, a later version of the Kainos assay protocol (from October 2010 onwards) includes an improved wash instruction. Using plasma samples from patients with renal impairment, Devaraj and co-workers also found good inter- and intra assay precision for cFGF-23 assay (CV between 4–10.5%), however, the CVs for iFGF-23 Immutopic assay were found to be poor (6–37.5%) [12].
Poor analytical agreement exists between the commercially available FGF-23 assays, due principally to the lack of a reference method. The performance of four commercially available methods [iFGF-23 assays from Immutopics, Kainos and Millipore (USA) and a cFGF-23 assay from Immutopics] were recently compared using plasma from 31 healthy adults and 36 patients undergoing hemodialysis [13]. A broad range of FGF-23 values were obtained: whereas the patient ranges fell between 154–2561 pg/mL and 447–2063 pg/mL for iFGF-23 and cFGF-23 assay respectively, the levels for healthy adults ranged from 9.9–62 pg/mL for the two assay types. Poor analytical agreement was observed between the assays particularly in the patient group. No agreement of test results was found between the iFGF-23 and cFGF-23 assays and this was more evident at physiological concentrations than in the haemodialysis group [13]. The lack of analytical agreement between these commercially available FGF-23 methods emphasizes that they cannot be used interchangeably and that a comparison of findings from different assays requires careful interpretation. The above evaluation study was performed with plasma samples [13]. A further consideration is some assays are restricted to the sample type that can be used. The iFGF-23 Kainos assay is suitable for both plasma and serum; however, the cFGF-23 Immutopics assay is established only for plasma [10, 13] providing lower or undetectable results in serum [10, 12]. Further method comparisons, ideally on larger numbers of samples and in different patient groups, would provide a valuable insight in this area and help identify which assay type is the most suitable for measuring circulating FGF-23. Nevertheless, studies measuring either intact or C-terminal FGF-23 have reported associations with mortality risk and decline in renal function [4, 14].
Stability of FGF-23: implication for large scale epidemiological studies
Limited evidence exists for the short-term stability of FGF-23 in collected blood samples (6 hours or less) and no information is available for its long-term stability in stored samples. Smith and co-workers investigated the short-term pre-analytical stability of FGF-23, measured using iFGF-23 and cFGF-23 ELISAs from Immutopics, by performing a number of timed experiments with blood taken from 15 patients with mild CKD [6]. The effect of aprotinin, a serine protease inhibitor, and a commercially available protease inhibitor cocktail to preserve FGF-23 after blood collection was also investigated [6]. In the absence of any preservative or inhibitor, iFGF-23 degraded by approximately 40% within 2 hours of collection even when the blood samples were separated into plasma. Conversely, 2 hours after blood collection the FGF-23 concentrations had increased by approximately 35% using the cFGF-23 assay. However, with the addition of the protease inhibitor cocktail the stability of both iFGF-23 and cFGF-23 in the samples extended up to 4 hours (less than 10% change). Based on this evidence, it appears that FGF-23 cannot be measured reliably in blood samples collected without the use of any preservative or inhibitors. This could be a serious limitation for large-scale epidemiological studies, particularly if samples have already been collected and are in storage and for blood collection methods that need to be simple in order to be cost-effective and feasible.
Preliminary findings from our laboratory using samples from 54 CKD patients suggest that FGF-23 (measured by both the iFGF-23 Kainos ELISA and the cFGF-23 Immutopics ELISA) remains stable in whole blood stored for up to 96 hours without the use of a preservative [15]. The apparent lack of agreement with the results from Smith et al. may be explained by differences in the methods of sample collection. Smith et al. used a single K2-EDTA blood tube for each participant which was re-sampled at each time-point whereas we collected separate blood tubes corresponding to each time-point.
Large-scale epidemiological studies often involve the long-term frozen storage of samples prior to biomarker analyses, particularly nested case-control designed studies where it may take several years for sufficient incident cases to materialize. Limited information is available to help understand the impact of long-term frozen storage on the stability of many biomarkers, including FGF-23. Studies investigating the stability of biomarkers in different sample types stored at various temperatures (for example, −40 °C, −80 °C and in liquid nitrogen vapour) will have immense value, particularly in support of long-term blood based prospective studies and biobanks.
Summary
FGF-23 is a key regulator of phosphate homeostasis and has emerged as an important biomarker in patients with CKD. Despite an increasing amount of literature, there are still unanswered questions related to FGF-23 sample stability and the availability of robust reliable methods for measuring FGF-23. Further studies into these areas will improve the quality of clinical research into the use of FGF-23 as a potential early biomarker in CKD.
References
1. ADHR Consortium. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet. 2000; 26: 345–348.
2. Wolf M. Forging forward with 10 burning questions on FGF23 in kidney disease. J Am Soc Nephrol. 2010; 21: 1427–1435.
3. Quarles LD. Role of FGF23 in vitamin D and phosphate metabolism: implications in chronic kidney disease. Exp Cell Res. 2012; 318: 1040–1048.
4. Isakova T, Xie H, Yang W, et al. Fibroblast growth factor 23 and risks of mortality and end-stage renal disease in patients with chronic kidney disease. JAMA 2011; 305: 2432–2439.
5. Russo D, Battaglia Y. Clinical significance of FGF-23 in patients with CKD. Int J Nephrol. 2011; 2011: 364890.
6. Smith ER, Ford ML, Tomlinson LA, Weaving G, Rocks BF, Rajkumar C, Holt SG. Instability of fibroblast growth factor-23 (FGF-23): implications for clinical studies. Clin Chim Acta 2011; 412: 1008–1011.
7. London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant. 2003; 18: 1731–1740.
8. Jean G, Terrat JC, Vanel T, Hurot JM, Lorriaux C, Mayor B, Chazot C. High levels of serum fibroblast growth factor (FGF)-23 are associated with increased mortality in long haemodialysis patients. Nephrol Dial Transplant. 2009; 24: 2792–2796.
9. Titan SM, Zatz R, Graciolli FG, dos Reis LM, Barros RT, Jorgetti V, Moyses RM. FGF-23 as a predictor of renal outcome in diabetic nephropathy. Clin J Am Soc Nephrol. 2011; 6: 241–247.
10. Shimada T, Urakawa I, Isakova T, Yamazaki Y, Epstein M, Wesseling-Perry K, Wolf M, Salusky IB, Jüppner H. Circulating fibroblast growth factor 23 in patients with end-stage renal disease treated by peritoneal dialysis is intact and biologically active. J Clin Endocrinol Metab. 2010; 95: 578–585.
11. Heijboer AC, Levitus M, Vervloet MG, Lips P, ter Wee, PM, Dijstelbloem HM, Blankenstein MA. Determination of fibroblast growth factor 23. Ann Clin Biochem. 2009; 46: 338–340.
12. Devaraj S, Duncan-Staley C, Jialal I. Evaluation of a method for fibroblast growth factor-23: a novel biomarker of adverse outcomes in patients with renal disease. Met Syndr Relat Disord. 2010; 8: 477–482.
13. Smith ER, McMahon LP, Holt SG. Method-specific differences in plasma fibroblast growth factor 23 measurement using four commercial ELISAs. Clin Chem Lab Med. 2013; 51: 1971–1981.
14. Fliser D, Kollerits B, Neyer U, Ankerst DP, Lhotta K, Lingenhel A, Ritz E, Kronenberg F, Kuen E, Konig P, Kraatz G, Mann JF, Muller GA, Kohler H, Riegler P. Fibroblast growth factor 23 (FGF23) predicts progression of chronic kidney disease: the Mild to Moderate Kidney Disease (MMKD) Study. J Am Soc Nephrol. 2007; 18: 2600–2608.
15. Illingworth N, Edmans M, Clark S, Kumar A, Sutherland S, Herrington W, Hill M. Investigation of FGF-23 sample & assay suitability for large scale epidemiological studies. Ann Clin Biochem. 2013; 50: 73–74
The authors
Aishwarya Kumar1* PhD; Will Herrington2 MBBS, MRCP; Sarah Clark1 PhD; and Michael Hill1 PhD
1Clinical Trial Service Unit and Epidemiological Studies Unit (CTSU), University of Oxford, Oxford, UK
2Oxford Kidney Unit, Oxford University Hospitals, Oxford, UK
*Corresponding author
E-mail: Aishwarya.Kumar@ctsu.ox.ac.uk
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by James D. Peele, PhD The HEp-2 immunofluorescence assay (IFA) for ANA screening is excellent for ruling out many connective tissue diseases, but a positive result seldom translates into a clinically meaningful diagnosis. A new automated, efficient, enzyme immunoassay for ANA screening provides reliable, objective information that can be applied clinically with confidence.
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