ARTICLE IMPACT STATEMENT ... September : JALM 1 Copyright 2017 by American Association for Clinical Chemistry.

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1 Renal Function Influences Diagnostic Markers in Serum and Urine: A Study of Guanidinoacetate, Creatine, Human Epididymis Protein 4, and Neutrophil Gelatinase Associated Lipocalin in Children Cathrin L. Salvador, 1,2 * Camilla Tøndel, 3,4 Alexander D. Rowe, 1 Anna Bjerre, 5 Atle Brun, 6,7 Damien Brackman, 3 Nils Bolstad, 1,2 and Lars Mørkrid 1,2 Background: Impaired renal function may affect the level of diagnostic disease markers. The aim of the study was to investigate the effect of measured glomerular filtration rate (GFR) on 4 diagnostic markers in blood and urine guanidinoacetate (GAA), creatine (CRE), human epididymis protein 4 (HE4), and neutrophil gelatinase associated lipocalin (NGAL) and how this could affect the decision and reference limits. Methods: We examined 96 children (median age 9.2 years, range ) with different stages of chronic kidney disease (CKD). GFR [median 65.9 ml min 1 (1.73 m 2 ) 1, range ] was measured by iohexol clearance using 7 venous blood samples after iohexol injection. Fasting serum and urinary GAA, CRE, HE4, NGAL, and creatinine (crn) were analyzed. After appropriate transformation of the markers, a multiple linear regression analysis examined the influence of age, sex, and measured GFR. Results: The level of GFR significantly affected S-GAA (P = ) and U-GAA/crn (P = ), leading to decreased values in renal impairment. GFR did not correlate significantly with the level of CRE and to a minor degree did the U-CRE/crn ratio (P = 0.54 and 0.01, respectively). The level of GFR significantly affected S-HE4 (P = ) and U-HE4/S-HE4 ratio (P = ) with increased serum values and decreased U-HE4/S-HE4 ratio in renal impairment. S-NGAL increased with decreasing kidney function (P = ). Conclusions: Diagnostic disease markers may be influenced by the renal function, and this must be taken into account when interpreting test results. Decreased renal function could change the level of the marker above or below decision limits, leading to diagnostic misinterpretation. Clinical Trial Registration: ClinicalTrials.gov, Identifier NCT , NCT ?term=tondel&rank=2 IMPACT STATEMENT We investigated the effect of measured GFR on 4 diagnostic markers in blood and urine and how this could affect the decision limits. We examined 96 children with different stages of chronic kidney disease. GFR was measured by iohexol clearance. Diagnostic disease markers were influenced by the renal function, and this must be taken into account when interpreting test results. Decreased renal function could change the level of the marker above or below decision limits leading to diagnostic misinterpretation. This study may also serve as a model to investigate other disease markers. September : JALM 1 Copyright 2017 by American Association for Clinical Chemistry.

2 GFR Influences Guanidinoacetate, HE4, and NGAL Renal function is impaired in many different patient groups, both as a consequence of a primary kidney disorder or secondary to treatment that influences glomerular filtration or tubular metabolism and transport (e.g., chemotherapy) (1). Molecules in plasma may be eliminated by glomerular filtration, tubular secretion, or both; hence, the total renal clearance will be of importance when evaluating different diagnostic markers in plasma or urine. Impaired renal function, preferably expressed by glomerular filtration rate (GFR), 8 may affect the level of these markers differently and therefore should be examined in each situation. Guanidinoacetate (GAA) and creatine (CRE) in blood and urine are essential diagnostic markers for creatine deficiency syndrome (2). Urinary GAA/ creatinine (crn) and CRE/crn are measured as screening in children with unexplained intellectual disability and delayed language development. In guanidinoacetate methyltransferase (GAMT) deficiency, the level of urinary GAA/crn ratio and blood GAA is increased and CRE/GAA ratio decreased. The opposite is seen in arginine:glycine amidinotransferase (AGAT) deficiency. X-linked creatine transporter (CRT) defect usually presents with increased CRE/crn ratio in urine, at least in males. Some studies have shown low serum concentration of GAA in patients with chronic kidney disease (CKD), especially in the lowest estimating GFR range (3, 4). To our knowledge, the consequences of variations in measured GFR (mgfr) on the levels of GAA and CRE in blood and urine have previously not been investigated in children. Human epididymis protein 4 (HE4) is a tumor marker for ovarian cancer, used either in combination with CA125 or alone. HE4 is not a marker commonly used in pediatric patients, but previous studies have demonstrated higher HE4 levels in blood in patients with high crn levels (5) and in patients with decreased renal function based on different GFR-estimating formulas (6, 7); hence, this is an example to illustrate this mechanism. As far as we know, the relationship between mgfr and HE4 has previously not been examined. Neutrophil gelatinase associated lipocalin (NGAL) is a protein that may be used for early detection of acute kidney injury, and the plasma and urinary levels of NGAL are thought to increase before plasma crn increases (8 10). NGAL is widely studied in both adults and children after cardiac surgery, but also in critically ill patients and after contrast agents (8, 11, 12). One study examined the relationship between serum NGAL and ioversol clearance in pediatric patients with CKD stages 2 4 and showed that serum NGAL outperforms estimated GFR (egfr) and cystatin C at lower levels of mgfr (13). To our knowledge, there are no publications that describe the relationship between mgfr (based on iohexol clearance) and NGAL in both urine and blood in children with CKD stages 1 5. The purpose of our study was to investigate the influence of kidney function, represented by measured GFR on the following 4 diagnostic disease 1 Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway; 2 Faculty of Medicine, Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 3 Department of Pediatrics, Haukeland University Hospital, Bergen, Norway; 4 Department of Clinical Medicine, University of Bergen, Bergen, Norway; 5 Department of Pediatrics, Oslo University Hospital, Oslo, Norway; 6 Laboratory for Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway; 7 Department of Clinical Science, University of Bergen, Bergen, Norway. *Address correspondence to this author at: Department of Medical Biochemistry, Oslo University Hospital, PB 4950 Nydalen, 0424 Oslo, Norway. Fax ; catsal@ous-hf.no. This study was previously presented as a poster at the SSIEM (Society for the Study of Inborn Errors of Metabolism) Congress in Rome, Italy, September DOI: /jalm American Association for Clinical Chemistry 8 Nonstandard abbreviations: GFR, glomerular filtration rate; GAA, guanidinoacetate; CRE, creatine; crn, creatinine; GAMT, guanidinoacetate methyltransferase; AGAT, arginine:glycine amidinotransferase; CRT, creatine transporter; CKD, chronic kidney disease; mgfr, measured GFR; HE4, human epididymis protein 4; NGAL, neutrophil gelatinase associated lipocalin; egfr, estimated GFR; I, iohexol; IDMS, isotope dilution mass spectrometry. 2 JALM :02 September 2017

3 GFR Influences Guanidinoacetate, HE4, and NGAL ARTICLE Table 1. Basic characteristics of the population. Value given as median (range) Variable or number Total number, F/M 96 (41/55) Age, years 9.2 ( ) Body weight, kg 28.3 ( ) Height, cm 134 (59 177) mgfr, 7-point iohexol clearance, ml min 1 (1.73 m 2 ) ( ) S-GAA, μmol/l 1.37 ( ) S-CRE, μmol/l 56.9 ( ) U-GAA/crn, μmol/mmol 30.1 ( ) U-CRE/crn, μmol/mmol 56.5 ( ) S-HE4, pmol/l 114 ( ) U-HE4/crn, pmol/mmol 937 ( ) U-HE4/S-HE4 37 ( ) S-NGAL, μg/l 114 (40 570) U-NGAL/crn, μg/mmol 6.1 ( ) markers in blood and urine, (a) GAA, (b) CRE, (c) HE4, and (d) NGAL, and how this could affect the decision and reference limits in a cohort of children with CKD. MATERIALS AND METHODS Study population Children (n = 96) at Haukeland University Hospital and Oslo University Hospital with CKD were recruited (ClinicalTrial.gov Identifier NCT ). The patients included had stable kidney function and a variety of kidney disorders, had no edema, and represented different GFR stages: 28, 27, 23, and 18 patients in CKD stage 1, 2, 3, and 4 5, respectively. The median age was 9.2 years (range 3 months to 17.5 years), and median mgfr was 65.9 ml min 1 (1.73 m 2 ) 1 (range ) (Table 1). Exclusion criteria were allergy to iohexol and x-ray contrast agents given less than 5 days before the survey. The study was approved by the Regional Ethics Committee of Western Norway, and written informed consent was obtained in all cases before inclusion in the study. The procedures followed were in accordance with the Helsinki Declaration and Good Clinical Practice. Methods We obtained a serum sample (3 ml) at baseline before the injection of iohexol (Omnipaque, 300 mg iodine/ml, GE Healthcare; i.e., 647 mg iohexol/ ml) for measurements of different biomarkers and to exclude the presence of potential substances interfering with the measurements of iohexol by HPLC. Omnipaque was administered via an intravenous cannula in doses according to the patients weight: <10 kg; 1 ml, kg; 2 ml, kg; 3 ml, kg; 4 ml, 40 kg; 5 ml. The syringe with iohexol was weighed before and after injection. The dose of iohexol (in mg) was calculated from the difference in syringe weight multiplied by the concentration of iohexol (647 mg/ml) and then divided by its density at room temperature (1.345 g/ml). The cannula was flushed with 15 ml physiologic saline. Serum samples (0.5 ml) were collected from the vein of the contralateral arm of the iohexol injection at 10, 30, 120, 180, 210, 240, and 300 min after injection of iohexol given the 7-time point reference GFR. In 29 of the 96 individuals, the second serum sample was drawn after 60 min instead of 30 min. Exact timing for each blood sampling was recorded. Serum concentrations of iohexol were analyzed by a HPLC system accredited by the Norwegian Accreditation and complies with the requirements of NS-EN ISO I5189, described elsewhere (14). HE4 in serum (S) and urine (U) was measured using the Elecsys HE4 assay on Cobas e601 (Roche Diagnostics) and NGAL (S and U) using Roche Modular P NGAL immunoassay (reagents from Gentian, Moss, Norway; Quantification of CRE and GAA (S and U) was performed according to Bodamer et al. (15). In short, isotopically labeled analogs of the analytes were added to a protein precipitation solution. After centrifuga- September : JALM 3

4 GFR Influences Guanidinoacetate, HE4, and NGAL Table 2. Estimated marker changes when mgfr decreases from 90 to 30 ml min 1 (1.73 m 2 ) 1 or age increases from 2 to 17 years. a Diagnostic marker Change of marker value when mgfr decreases from 90 to 30 ml min 1 (1.73 m 2 ) 1 Exceeding Gowans' criterion, yes/no Change of marker value when age increases from 2 to 17 years Exceeding Gowans' criterion, yes/no Optimal λ S-GAA, μmol/l Yes Yes Reference interval from the literature (reference) <15 years: ; >15 years: (33) S-CRE, μmol/l No Yes <10 years: >10 years: 6 50 (33) U-GAA/crn, μmol/mmol Yes No Continuous reference limits (21) U-CRE/crn, μmol/mmol Yes Yes Continuous reference limits (21) S-HE4, pmol/l Yes Yes years: years: years: <60 (5, 34) U-HE4/S-HE Yes Yes Not known S-NGAL, μg/l Yes Yes Cutoff > μg/l (12, 35, 36) U-NGAL/crn, μg/mmol Yes Yes Age-specific normative range: years: years: years: years: 15.7 Cutoff > μg/l (12, 36, 37) a Baseline values, to the left in columns 2 and 4, are age-adjusted upper-reference limits. The new marker values, to the right, result from regression analyses. Optimal Box-Cox λ values, and whether a change exceeds Gowans' criterion or not, are also presented. tion, reagents for derivitizing the analytes to butyl esters was added to the samples, followed by LC-MS/MS. Creatinine (S and U) was measured with an enzymatic calorimetric method (reagents from Roche Diagnostics), isotope dilution mass spectrometry (IDMS) traceable. The laboratories participate in several external quality programs: LabQuality (crn, HE4), Equalis (iohexol clearance), and European Research Network for evaluation and improvement of screening, Diagnosis, and treatment of Inherited Metabolic disorders (ERNDIM) (CRE and GAA). Calculation of GFR The reference method for GFR in this study was based on the 7 time point (10, 30/60, 120, 180, 210, 240, 300 min) iohexol clearance and calculated by the method of Sapirstein (16, 17). GFR was normalized to body surface area calculated by the method of Haycock et al. (18). Statistical analysis All variables were Box-Cox transformed to remove skewness. Optimal λ value for mgfr was 1. Optimal λ values for CRE, GAA, HE4, and NGAL in urine and serum are presented in Table 2 and were based on a maximal correlation coefficient in the central 95% variation interval of the z-score plot between the transformed variable (abscissa) and its corresponding z-score (ordinate). A z-score was calculated from the rank of sorted values (k) via its corresponding percentile, p =(k 3/8)/(n + 4 JALM :02 September 2017

5 GFR Influences Guanidinoacetate, HE4, and NGAL ARTICLE 1/4) in the standard normal distribution (19). No substantial residual kurtosis was observed in the individual z-score plots. An optimal λ value for the covariate age was determined to give its distribution a maximal SD/range ratio because this minimizes the effect of high-leverage points. The biological significance of a statistically significant effect size was evaluated against Gowans' criterion (effect size <0.25 total biological variation) (Table 2) (20). Multiple forward and backward linear regression analysis was then performed between the Box-Cox transformed diagnostic marker (dependent variable) and the following covariates: age, sex, and mgfr. P value for inclusion and exclusion of variables was Residuals were converted to z-scores and examined for normal distribution. To evaluate the influence of renal function on the diagnostic markers, the difference in Box-Cox transformed values for the difference in mgfr values was calculated and finally retransformed. The Pearson correlation coefficient was used. Microsoft Excel (version 2010) and IBM SPSS Statistics Package (version 21) were used for statistical analysis. RESULTS Characteristics of the participants (n = 96) are presented in Table 1. Table 2 shows the effect of a change (Δ) in mgfr and age on the level of a given value of the diagnostic disease marker. GFR had a significant affect on S-GAA (P = ) and U-GAA/crn (P = ), leading to decreased values in renal impairment (Fig. 1, A and C). In contrast, S-CRE did not correlate significantly with GFR and only to a minor degree did the U- CRE/crn ratio (P = 0.54 and 0.01, respectively) (Fig. 1, B and D). A change in GFR from, for example, 100 to 10 ml min 1 (1.73 m 2 ) 1, reduced the expected S-GAA from 2 to 1.5 μmol/l and thus a modest change could alter the value well below the lower reference limit. The effect of mgfr on U-GAA/crn was even more pronounced, and the U-GAA/crn level could easily be shifted below the diagnostic limit for GAMT deficiency screening and, in addition, could result in false-positive results for AGAT deficiency (Table 2 and Fig. 2A). The same effect was not observed for screening of a CRT deficiency (Fig. 2B). Age strongly influenced the level of S-GAA, S-CRE, and U-CRE/crn (P < 0.001), but not U-GAA/crn in this patient group (P = 0.50) [Table 2 and Supplemental Figs. 1 4 (see the Data Supplement that accompanies the online version of this article at vol2/issue2)]. Statistically, the U-GAA/crn ratio also differed with respect to sex, with lower values in males (P = 0.003) (see Fig. 3 in the online Data Supplement). The difference was also shown to be biologically relevant (exceeding the Gowans' criterion). Sex did not influence S-GAA (P = 0.42), S-CRE (P = 0.34), or U-CRE/crn (P = 0.17) to the same extent (see Figs. 1, 2, and 4 in the online Data Supplement). The level of GFR significantly affected the S-HE4 (P = ) and U-HE4/S-HE4 ratio (P = ), such that patients with low GFR showed increased serum values and a decreased U-HE4/S- HE4 ratio (Table 2, Fig. 3, A and B). Even a moderate decrease in GFR could increase the level of the S-HE4 above the reference limits regardless of age. Age and sex did not influence the U-HE4/S-HE4 ratio significantly (P = 0.15, just above Gowens' criterion), and there was no significant sex difference for S-HE4 (P = 0.08), but a minor age effect (P = 0.035) was observed (see Figs. 5 and 6 in the online Data Supplement). NGAL in serum and urine was also affected by GFR. We found a considerable increase in S-NGAL levels with decreasing kidney function (P = ) and to a smaller extent an increase in U- NGAL/crn (P = 0.045) (Table 2, Figs. 4A and 4B). Age influenced the marker in both serum and urine (P = and 0.012, respectively), but there was no significant sex difference (P = 0.11 and 0.10, respectively) (see Figs. 7 and 8 in the online Data Supplement). September : JALM 5

6 GFR Influences Guanidinoacetate, HE4, and NGAL Fig. 1. Scatter plots of S-GAA, S-CRE, U-GAA/crn, U-CRE/crn, and mgfr in children. (A), Correlation between untransformed S-GAA values and mgfr in children with CKD (n = 96); the gray area is the CI around the regression line; correlation coefficient, r = (B), Correlation between untransformed S-CRE values and mgfr in children with CKD (n = 96); the gray area is the CI around the regression line; correlation coefficient, r = (C), Correlation between z-score U-GAA/crn and mgfr in children with CKD (n = 96) (quadratic line fit used in the figure); the gray area is the CI around the regression line; correlation coefficient, r = (D), Correlation between z-score U-CRE/crn and mgfr in children with CKD (n = 96); the gray area is the CI around the regression line; correlation coefficient, r = DISCUSSION The present study showed that the majority of the diagnostic disease markers in blood and urine investigated in this cohort were influenced by the kidney function. U-GAA/crn level could easily be shifted below the diagnostic limit for screening of GAMT deficiency. A small change in GFR could increase the level of the S-HE4 above the reference limit regardless of age. A considerable increase in S-NGAL levels was observed with decreasing kidney function. The ratio U-CRE/crn is strongly dependent on age and U-GAA/crn on sex, with higher urine markers seen in females; however, Gowans' criterion suggests that combined reference limits were adequate for both sexes in children (21). In our cohort, the U-GAA/crn ratio was higher in females than males, but sex did not influence the U-CRE/ crn ratio. We found no age effect on the U-GAA/crn ratio in this cohort. Studies on guanidine compounds in rats (22, 23) have demonstrated lower serum GAA levels in renal injury. This result might be explained by reduced AGAT activity, the enzyme 6 JALM :02 September 2017

7 GFR Influences Guanidinoacetate, HE4, and NGAL ARTICLE Fig. 2. Percentile charts for U-GAA/crn and U-CRE/crn. (A), Percentile charts for U-GAA/crn [Morkrid et al. (21)]. Dotted lines indicate, from bottom to top, 2.5th and 97.5th percentile. Dashed line indicates the median. Square symbols represent the patients with mgfr <30 (n = 18), triangular symbols mgfr (n = 23), diamond symbols mgfr (n = 27), and circular symbols mgfr 90 ml min 1 (1.73 m 2 ) 1 (n = 28). (B), Percentile charts for U-CRE/crn [Morkrid et al. (21)]. Dotted lines indicate, from bottom to top, 2.5th and 97.5th percentile. Dashed line indicates the median. Square symbols represent the patients with mgfr <30 (n = 18), triangular symbols mgfr (n = 23), diamond symbols mgfr (n = 27), and circular symbols mgfr 90 ml min 1 (1.73 m 2 ) 1 (n = 28). that catalyzes conversion of glycine and arginine to ornithine and GAA (24). The reduction of AGAT activity in the renal tubules has also been confirmed in rabbits with chronic renal failure (CRF) (25), and some adult patients with CRF showed low serum and urine GAA (3, 4). This is in agreement with our study. GFR did not influence creatine in the same way, and one may speculate that this may be due to a compensatory elevation of GAMT activity in other organs, e.g., pancreas and liver (26). Several biological and lifestyle factors affect HE4 concentration (27). One study found that high serum HE4 concentrations were associated with high serum crn, but it was uncertain whether the reduction in GFR with increasing age could explain the increased HE4 in the elderly patients (5). Other studies have demonstrated a significant inverse relation between HE4 concentration and estimated GFR (6, 7), and therefore HE4 results should be interpreted with caution in both patients with renal disorders and in individuals with declining GFR as a consequence of treatment with chemotherapy or in the elderly (1). Kappelmayer et al. (28) proposed an algorithm for predicting ovarian cancer in patients with kidney disorders using serum HE4 and CA125 levels corrected for egfr. If HE4 was >147 pmol/l and egfr was >48 ml min 1 (1.73 m 2 ) 1, the chance of a tumor was estimated to be 95%, but in postmenopausal women with HE4 levels >210 pmol/l and egfr <28 ml min 1 (1.73 m 2 ) 1, the chance of a tumor was estimated at 30%. Our study demonstrated that a moderate change in mgfr may increase the serum HE4 levels above the reference limit. This result is not a marker commonly used in pediatric patients, but the purpose was to investigate the effect of mgfr on the marker and illustrate how this could affect the general interpretation of the marker. There are several studies on NGAL in different patient populations (8, 12). Although NGAL concentrations are known to increase with a reduction in GFR, it is also a marker of tubular damage, and thus it is not only dependent on the GFR (29, 30). September : JALM 7

8 GFR Influences Guanidinoacetate, HE4, and NGAL Fig. 3. Scatter plots of S-HE4 and U-HE4/S-HE4 ratio and mgfr in children. (A), Correlation between untransformed S-HE4 values and mgfr in children with CKD (n = 96) (quadratic line fit used in the figure); the gray area is the CI around the regression line; correlation coefficient, r = (B), Correlation between untransformed U-HE4/S-HE4 ratio and mgfr in children with CKD (n = 96) (quadratic line fit used in the figure); the gray area is the CI around the regression line; correlation coefficient, r = JALM :02 September 2017

9 GFR Influences Guanidinoacetate, HE4, and NGAL ARTICLE Fig. 4. Scatter plots of S-NGAL and U-NGAL/crn and mgfr in children. (A), Correlation between untransformed S-NGAL values and mgfr in children with CKD (n = 96) (quadratic line fit used in the figure); the gray area is the CI around the regression line; correlation coefficient, r = (B), Correlation between untransformed U-NGAL/crn values and mgfr in children with CKD (n = 96); the gray area is the CI around the regression line; correlation coefficient, r = September : JALM 9

10 GFR Influences Guanidinoacetate, HE4, and NGAL Serum NGAL investigated in children with CKD stages 2 4 has shown that with GFR <30 ml min 1 (1.73 m 2 ) 1, serum NGAL correlated inversely with mgfr (ioversol clearance) (r = 0.62). This study, where ioversol injection with 3 sampling times and an in-house ELISA was used to assess GFR, indicated that serum NGAL may be an additional measure of kidney dysfunction in CKD (12). Another study showed that plasma NGAL increased progressively with GFR reduction in adult patients with stage 2 CKD, generating a high number of false-positive diagnoses of acute kidney injury in stable CKD patients. The impairment of GFR affected plasma NGAL to a greater extent than urinary NGAL (30). Some authors have reported that urinary NGAL level is a better biomarker for chronic renal disease in children than serum NGAL, but in these studies, GFR was estimated and not measured accurately. Urinary NGAL level was increased in children with CKD with normal GFR when there was tubular dysfunction, as well as in patients with low GFR (31). Our study demonstrated that GFR influenced the serum NGAL level significantly more than urinary NGAL/crn ratio, and it could be difficult to interpret the NGAL values for acute kidney injury diagnosis in patients with CKD. The strength of our study was use of a highquality, 7-point iohexol clearance procedure for assessment of measured GFR. This methodology enabled use of values closer to the true GFR instead of less accurate GFR estimates based on crn and egfr formulas. Multipoint iohexol clearance is considered to be in good agreement with the gold standard inulin clearance (32). A limitation to our study is the number of patients (n = 96). On the other hand, few other studies had multipoint mgfr data in such a large cohort of children with CKD. Also, we had only 1 patient younger than 1 year, whereas 9 patients were <2 years. In conclusion, our results indicate that several diagnostic disease markers are influenced by renal function and that this must be taken into account when interpreting test results. Decreased renal function could change the level markers above or below decision limits and lead to incorrect diagnostic interpretation. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: C.L. Salvador, Health Trust of Western Norway, The Norwegian Society of Nephrology, Haukeland, and Oslo University Hospital. The laboratory at Oslo University Hospital was supported with NGAL reagents from Gentian, Moss, Norway. Expert Testimony: None declared. Patents: None declared. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. Acknowledgments: The authors express gratitude to the pediatric study nurses Mai Britt Lynum, Hildur Grindheim, and Renathe Håpoldøy for technical assistance with the sample collection; to the laboratory engineers Kjersti Carstensen (iohexol analyses), Linda Langdahl (HE4 analyses), Astrid Steiro, and Hildur Tannum (NGAL analyses); and the National Unit of Laboratory Diagnostic for Inborn Errors of Metabolism (CRE and GAA analyses). REFERENCES 1. de Jonge MJ, Verweij J. Renal toxicities of chemotherapy. Semin Oncol 2006;33: Mercimek-Mahmutoglu S, Salomons GS. Creatine deficiency syndromes. GeneReviews, Seattle (WA): University of Washington; Marescau B, Nagels G, Possemiers I, De Broe ME, Becaus.. 10 JALM :02 September 2017

11 GFR Influences Guanidinoacetate, HE4, and NGAL ARTICLE I, Billiouw JM, et al. Guanidino compounds in serum and urine of nondialyzed patients with chronic renal insufficiency. Metabolism 1997;46: Torremans A, Marescau B, Kranzlin B, Gretz N, Billiouw JM, Vanholder R, et al. Biochemical validation of a rat model for polycystic kidney disease: comparison of guanidino compound profile with the human condition. Kidney Int 2006;69: Bolstad N, Oijordsbakken M, Nustad K, Bjerner J. Human epididymis protein 4 reference limits and natural variation in a Nordic reference population. Tumour Biol 2012;33: Ferraro S, Pasqualetti S, Carnevale A, Panteghini M. Cystatin c provides a better estimate of the effect of glomerular filtration rate on serum human epididymis protein 4 concentrations. Clin Chem Lab Med 2016;54: Nagy B, Jr., Krasznai ZT, Balla H, Csoban M, Antal-Szalmas P, Hernadi Z, Kappelmayer J. Elevated human epididymis protein 4 concentrations in chronic kidney disease. Ann Clin Biochem 2012;49: Singer E, Marko L, Paragas N, Barasch J, Dragun D, Muller DN, et al. Neutrophil gelatinase-associated lipocalin: pathophysiology and clinical applications. Acta Physiol (Oxf.) 2013;207: Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 2003;14: Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 2005;365: Benzer M, Alpay H, Baykan O, Erdem A, Demir IH. Serum ngal, cystatin c and urinary nag measurements for early diagnosis of contrast-induced nephropathy in children. Ren Fail 2016;38: Haase-Fielitz A, Haase M, Devarajan P. Neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury: a critical evaluation of current status. Ann Clin Biochem 2014;51: Mitsnefes MM, Kathman TS, Mishra J, Kartal J, Khoury PR, Nickolas TL, et al. Serum neutrophil gelatinaseassociated lipocalin as a marker of renal function in children with chronic kidney disease. Pediatr Nephrol 2007;22: T øndel C, Bolann B, Salvador CL, Brackman D, Bjerre A, Svarstad E, Brun A. Iohexol plasma clearance in children: validation of multiple formulas and two-point sampling times. Pediatr Nephrol 2016;32: Bodamer OA, Bloesch SM, Gregg AR, Stockler-Ipsiroglu S, O'Brien WE. Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry. Clin Chim Acta 2001;308: Sapirstein LA, Vidt DG, Mandel MJ, Hanusek G. Volumes of distribution and clearances of intravenously injected creatinine in the dog. Am J Physiol 1955;181: Schwartz GJ, Furth S, Cole SR, Warady B, Munoz A. Glomerular filtration rate via plasma iohexol disappearance: pilot study for chronic kidney disease in children. Kidney Int 2006;69: Haycock GB, Schwartz GJ, Wisotsky DH. Geometric method for measuring body surface area: a heightweight formula validated in infants, children, and adults. J Pediatr 1978;93: Altman D. Practical statistics for medical research. Boca Raton (FL): Chapman & Hall/CRC Texts in Statistical Science; Gowans EM, Hyltoft Petersen P, Blaabjerg O, Horder M. Analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area. Scand J Clin Lab Invest 1988;48: Morkrid L, Rowe AD, Elgstoen KB, Olesen JH, Ruijter G, Hall PL, et al. Continuous age- and sex-adjusted reference intervals of urinary markers for cerebral creatine deficiency syndromes: a novel approach to the definition of reference intervals. Clin Chem 2015;61: Ozasa H, Watanabe T, Nakamura K, Fukunaga Y, Ienaga K, Hagiwara K. Changes in serum levels of creatol and methylguanidine in renal injury induced by lipid peroxide produced by vitamin e deficiency and gsh depletion in rats. Nephron 1997;75: Levillain O, Marescau B, de Deyn PP. Guanidino compound metabolism in rats subjected to 20% to 90% nephrectomy. Kidney Int 1995;47: Edison EE, Brosnan ME, Meyer C, Brosnan JT. Creatine synthesis: production of guanidinoacetate by the rat and human kidney in vivo. Am J Physiol Renal Physiol 2007; 293:F Kuroda M. Study on impaired metabolism of guanidinoacetic acid in chronic renal failure rabbits with special reference to impaired conversion of arginine to guanidinoacetic acid. Nephron 1993;65: Walker JB. Creatine: biosynthesis, regulation, and function. Adv Enzymol Relat Areas Mol Biol 1979;50: Ferraro S, Schiumarini D, Panteghini M. Human epididymis protein 4: factors of variation. Clin Chim Acta 2015;438: Kappelmayer J, Antal-Szalmas P, Nagy B, Jr. Human epididymis protein 4 (HE4) in laboratory medicine and an algorithm in renal disorders. Clin Chim Acta 2015;438: Mori K, Nakao K. Neutrophil gelatinase-associated lipocalin as the real-time indicator of active kidney damage. Kidney Int 2007;71: Donadio C. Effect of glomerular filtration rate impairment on diagnostic performance of neutrophil gelatinaseassociated lipocalin and B-type natriuretic peptide as markers of acute cardiac and renal failure in chronic kidney disease patients. Crit Care 2014;18:R Nishida M, Kawakatsu H, Okumura Y, Hamaoka K. Serum and urinary neutrophil gelatinase-associated lipocalin levels in children with chronic renal diseases. Pediatr Int 2010;52: September : JALM 11

12 GFR Influences Guanidinoacetate, HE4, and NGAL 32. Schwartz GJ, Work DF. Measurement and estimation of gfr in children and adolescents. Clin J Am Soc Nephrol 2009;4: Verhoeven NM, Salomons GS, Jakobs C. Laboratory diagnosis of defects of creatine biosynthesis and transport. Clin Chim Acta 2005;361: Bevilacqua V, Chan MK, Chen Y, Armbruster D, Schodin B, Adeli K. Pediatric population reference value distributions for cancer biomarkers and covariatestratified reference intervals in the caliper cohort. Clin Chem 2014;60: Dent CL, Ma Q, Dastrala S, Bennett M, Mitsnefes MM, Barasch J, Devarajan P. Plasma neutrophil gelatinaseassociated lipocalin predicts acute kidney injury, morbidity and mortality after pediatric cardiac surgery: a prospective uncontrolled cohort study. Crit Care 2007; 11:R Haase M, Bellomo R, Devarajan P, Schlattmann P, Haase- Fielitz A. Accuracy of neutrophil gelatinase-associated lipocalin (NGAL) in diagnosis and prognosis in acute kidney injury: a systematic review and meta-analysis. Am J Kidney Dis 2009;54: Rybi-Szuminska A, Wasilewska A, Litwin M, Kulaga Z, Szuminski M. Paediatric normative data for urine NGAL/creatinine ratio. Acta Paediatr 2013;102: e JALM :02 September 2017