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Background: The Schwartz 2009 creatinine-based revised formula is the only pediatric GFR estimating formula, which is compatible with the recent global creatinine standardization. This formula is only applicable if enzymatic creatinine methods are used. We propose an equation, taking into account the relative bias caused by serum proteins to use Jaffe based creatinine data for GFR estimation., Methods: In a cohort study of 100 pediatric patients, serum creatinine was measured using a kinetic rate-blanked Jaffe assay (modified kinetic alkaline picrate method), a kinetic rate-blanked Jaffe compensated assay for reactive proteins and an enzymatic assay (creatinine plus method). Serum total protein, albumin, urea, uric acid and total bilirubin were measured with the use of commercial agents., Results: The difference in serum creatinine between the enzymatic method and the compensated Jaffe method was mainly dependent on the total protein concentration in serum (r(2)=0.61, p<0.001). After applying the proposed protein correction, corrected compensated Jaffe results and creatinine clearance values became interchangeable with enzymatic serum creatinine results (r(2)=0.99, p<0.001; Deming regression: slope: 0.9787, intercept: -0.351) and with the newly proposed Schwartz formula, respectively (r(2)=0.99, p<0.001; Deming regression: slope 1.004, intercept: 2.16)., Conclusions: In this study, we demonstrated the usability of the alkaline picrate method in the Schwartz formula, taking into account the relative bias caused by serum proteins.

It has been proposed that neonatal thyroid-stimulating hormone (TSH) concentrations are a good indicator of iodine deficiency in the population. A frequency of neonatal TSH concentrations above 5 mU/L below 3% has been proposed as the threshold indicating iodine sufficiency. The objective of the present study was to evaluate feasibility and usefulness of nation-wide neonatal TSH concentration screening results to assess iodine status in Belgium. All newborns born in Belgium during the period 2009-2011 (n = 377713) were included in the study, except those suffering from congenital hypothyroidism and premature neonates. The frequency of neonatal TSH concentrations above 5 mU/L from 2009 to 2011 in Belgium fluctuated between 2.6 and 3.3% in the centres using the same TSH assay. There was a significant inverse association between neonatal TSH level and birth weight. The longer the duration between birth and screening, the lower the TSH level. Neonatal TSH levels were significantly lower in winter than in spring or autumn and significantly lower in spring and summer than in autumn while significantly higher in spring compared to summer. In conclusion, despite that pregnant women in Belgium are mildly iodine deficient, the frequency of neonatal TSH concentrations above 5 mU/L was very low, suggesting that the neonatal TSH threshold proposed for detecting iodine deficiency needs to be re-evaluated. Although neonatal TSH is useful to detect severe iodine deficiency, it should not be recommended presently for the evaluation of iodine status in mildly iodine deficient regions.

Dried blood spot (DBS) methods are currently available for identification of a range of lysosomal storage disorders (LSDs). These disorders are generally characterized by a deficiency of activity of a lysosomal enzyme and by a broad spectrum of phenotypes. Diagnosis of LSD patients is often delayed, which is of particular concern as therapeutic outcomes (e.g. enzyme replacement therapy) are generally more favorable in early disease stages. Experts in the field of LSDs diagnostics and screening programs convened and reviewed experiences with the use of DBS methods, and discuss the diagnostic challenges, possible applications and quality programs in this paper. Given the easy sampling and shipping and stability of samples, DBS has evident advantages over other laboratory methods and can be particularly helpful in the early identification of affected LSD patients through neonatal screening, high-risk population screening or family screening., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Citrulline is considered to be a marker of absorptive enterocyte mass. Citrulline levels can be measured in plasma or dried blood spot (DBS) samples. The purpose of this study is to calculate reference intervals for plasma and DBS citrulline concentrations in children and to examine the effect of age and gender., Methods: In 151 healthy subjects ranging from 1 month to 20 years of age, plasma and DBS citrulline concentration were determined by using Liquid Chromatography-tandem Mass Spectrometry. Citrulline concentrations were examined in relation to age and gender. Reference values were calculated according to the guidelines of the International Federation of Clinical Chemistry and the National Committee on Clinical Laboratory Standards., Results: No significant influence of age and gender could be discerned on plasma or DBS citrulline concentration. In children, the reference intervals for citrulline bounded by the 2.5 and 97.5 percentiles are 13.31-69.05 μmol/L and 23.70-49.04 μmol/L for plasma and DBS samples respectively., Conclusions: The reference intervals for citrulline levels in healthy children are widely dispersed. Measuring citrulline concentrations in dried blood spots delivers no additional value to plasma measurements for the calculation of reference intervals in children., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The effect of dietary oligofructose and inulin supplementation on glucose metabolism in obese and non-obese cats was assessed. Two diets were tested in a crossover design; a control diet high in protein (46 % on DM basis), moderate in fat (15 %), low in carbohydrates (27 %), but no soluble fibres added; and a prebiotic diet, with 2.5 % of a mixture of oligofructose and inulin added to the control diet. Eight non-obese and eight obese cats were allotted to each of two diets in random order at intervals of 4 weeks. At the end of each testing period, intravenous glucose tolerance tests were performed. Area under the glucose curve (AUCgluc) was increased (P = 0.022) and the second insulin peak was delayed (P = 0.009) in obese compared to non-obese cats. Diets did not affect fasting plasma glucose concentrations, blood glucose response at each glucose time-point after glucose administration, AUCgluc, fasting serum insulin concentrations, area under the insulin curve, and height and appearance time of insulin response. Yet, analysis of acylcarnitines revealed higher propionylcarnitine concentrations (P = 0.03) when fed the prebiotic diet, suggesting colonic fermentation and propionate absorption. Prebiotic supplementation reduced methylmalonylcarnitine (P = 0.072) and aspartate aminotransferase concentrations (P = 0.025), both indicating reduced gluconeogenesis from amino acids. This trial evidenced impaired glucose tolerance and altered insulin response to glucose administration in obese compared to non-obese cats, regardless of dietary intervention; yet modulation of glucose metabolism by enhancing gluconeogenesis from propionate and inhibition of amino acid catabolism can be suggested.

Background: Anderson-Fabry disease (AFD) is an X-linked condition originating from a deficiency in alpha-galactosidase, a lysosomal enzyme. Multi-organ involvement ensues in early adulthood and vital organs are affected: the kidneys, brain, heart. Several reports however suggest that AFD is underdiagnosed., Methods: We screened a kidney transplant population using a two-tier approach. The first tier was the determination of alpha-galactosidase A (AGALA) activity using a dried blood spot on filter paper (DBFP); in the second tier, patients with the lowest alpha-galactosidase levels were further subjected to mutation analysis of the GLA gene., Results: From the database of 2328 patients, 1233 subjects met the inclusion criteria. Finally, after informed consent, 673 patients were screened (54.5%-395 women and 278 men). DBFP analysis resulted in a mean AGALA of 2.63 +/- 2.48 micromol/L/h (2.5 and 97.5 percentile were 0.0001 and 5.07 micromol/L/h, respectively). Eleven patients were subjected to further genetic analysis. In a male patient a pathogenic missense mutation p.Ala143Thr (c.427A>G) was identified., Conclusions: Our results show that the proposed approach can detect AFD patients in a until now seldomly screened high-risk group: kidney transplant patients. We conclude that screening for AFD in high-risk populations is a cost-effective, technically feasible and clinically valuable objective.

Background: The European In Vitro Diagnostics (IVD) directive requires traceability to reference methods and materials of analytes. It is a task of the profession to verify the trueness of results and IVD compatibility., Methods: The results of a trueness verification study by the European Communities Confederation of Clinical Chemistry (EC4) working group on creatinine standardization are described, in which 189 European laboratories analyzed serum creatinine in a commutable serum-based material, using analytical systems from seven companies. Values were targeted using isotope dilution gas chromatography/mass spectrometry. Results were tested on their compliance to a set of three criteria: trueness, i.e., no significant bias relative to the target value, between-laboratory variation and within-laboratory variation relative to the maximum allowable error., Results: For the lower and intermediate level, values differed significantly from the target value in the Jaffe and the dry chemistry methods. At the high level, dry chemistry yielded higher results. Between-laboratory coefficients of variation ranged from 4.37% to 8.74%. Total error budget was mainly consumed by the bias. Non-compensated Jaffe methods largely exceeded the total error budget. Best results were obtained for the enzymatic method. The dry chemistry method consumed a large part of its error budget due to calibration bias., Conclusions: Despite the European IVD directive and the growing needs for creatinine standardization, an unacceptable inter-laboratory variation was observed, which was mainly due to calibration differences. The calibration variation has major clinical consequences, in particular in pediatrics, where reference ranges for serum and plasma creatinine are low, and in the estimation of glomerular filtration rate.

Introduction: Fabry's disease (AFD) is an X-linked lysosomal storage disease, resulting from a deficiency in alpha-galactosidase A (AGALA). Untreated, this leads to precocious failure of vital organ function and death. As enzyme replacement therapy is available, it is of vital importance that affected individuals can be traced., Materials and Methods: We set up a screening in the Flemish haemodialysis population using a two-tier approach. The first tier was a determination of alpha-galactosidase A activity using a dried blood spot on filter paper, in the second tier, patients with the lowest alpha-galactosidase levels were further subjected to mutation analysis of the GLA gene., Results: 1284 patients (1047 women, 237 men) were evaluated for inclusion, eliminating patients with definite renal diagnoses. Total 922 patients (71.8 %) were screened (742 women, 180 men). Fifty seven patients were subjected to further genetic analysis. Three GLA mutation carriers were identified: two apparently nonrelated female patients carry the missense mutation p.Ala143Thr (c.427G > A), a missense mutation p.Trp236Arg (c.706T > C) was identified in a man. While the male patient had been clinically diagnosed with AFD, the female patients had remained unrecognized. Additional family based screening resulted in the identification of nine mutation carriers (four males and five females)., Discussion: We demonstrated that the prevalence of GLA mutation carriers in our haemodialysis population is 0.3%. Our results show that the proposed approach accurately detects AFD patients. We conclude that screening for AFD in high risk populations is a cost-effective, technically feasible and clinically valuable objective.

Background: Plasma Citrulline concentration has been correlated to functional enterocyte mass. Dried blood spot (DBS) analysis using LC-MSMS reduces sample amount needed. We optimized DBS elution to increase precision and accuracy of DBS LC-MSMS analysis., Method: DBS control samples were eluted in varying pH (2.2-7.0) and for varying times (15-75 min) and Cit, Arg and Orn were analyzed using LC-MSMS, with and without derivatization. In 20 volunteers, the DBS LC-MSMS assay was correlated with a plasma ion exchange HPLC method., Results: For Citrulline an extraction optimum was obtained at pH 2.6, whereas lower Arginine concentrations were found using low extraction pH. Increasing elution times lead to increased concentrations. Within-run CV was higher with, compared to without derivatization. No close association could be found between plasma HPLC and DBS LC-MSMS concentrations., Conclusion: Analysis of amino acids on DBS using LC-MSMS should be optimized regarding the purpose of the assay. In our study, most optimal results were obtained without derivatization and elution in pH 2.6 for 45 min. Cellular amino acids in DBS might influence the correlation of Cit with severity of enteral disorders. Therefore, further evaluation of DBS Cit as a marker for enteral disorders is warranted.

Background: Recently, automated urine test strip readers became available that can report quantitative data. We explored the possibility of measuring all ketone bodies (acetone, acetoacetate, 3-hydroxybutyrate) in urine with these test strips. Monitoring urinary ketone concentrations could offer the advantages of measuring higher values (due to the low renal thresholds) and being less sensitive to fluctuations., Methods: We evaluated URISYS 2400 (Roche) quantitative reflectance data for the ketone reflectance field and compared it with biochemical data from urine samples. Using an easy sample pre-treatment with 3-hydroxybutyrate dehydrogenase, we were able to assay 3-hydroxybutyrate as well, which normally does not react on urine test strips., Results: Within- and between-run reproducibility of the reflectance signal for high- and low-concentration urine pools was 11.0-3.6% and 11.0-5.8% for aceto-acetate, 8.2-9.2% and 10.4-16.1% for acetone, and 5.1-3.0% and 5.6-3.5% for 3-hydroxybutyrate, respectively. The lower limit of detection for acetoacetate was 0.13 mmol/L (CV=3.6%). Fair agreement was obtained between test strip data for ketones andcolorimetrically determined acetoacetate values (r=0.90)., Conclusions: In urine test strip analysis, quantitative ketone reflectance data allow a simple and fast analysis, offering affordable screening for the detection of ketone body production in diabetes, especially in emergency settings.

The quantification of monoclonal immunoglobulins by protein electrophoresis has been helpful in determining the prognosis for the patient. In a 65-year old man with documented IgG lambda monoclonal gammopathy, a discrepancy in the detection and quantification of this M-protein was found when studied by four different electrophoretic methods. On a high-resolution agarose method with acid violet staining a prominent peak was seen in the fast gamma-region. A lower resolution agarose method using an amido black staining showed a small mid-gamma restriction, and no spike was detected by high-resolution or lower resolution capillary zone electrophoresis. The discrepancy was not attributable to a migration in the beta-region leading to masking by transferrin or C3 peak, incorrect separation on CZE due to a high isoelectric point (pI > 8.5) or pentamerization of IgM M-proteins. Therefore, other causes for discrepancies should be investigated.

Background: The understanding of iron metabolism has increased substantially during the last decade. Several new transporters and iron regulating molecules have been described. Hepcidin, a small hepatic peptide has recently been proposed as a central mediator of dietary iron absorption. We have investigated the relationship between prohepcidin, the prohormone of hepcidin, and renal function and iron status., Methods: Forty six patients, referred for 51Cr-EDTA clearance were included in this study. Renal function was assessed by determination of serum creatinine, creatinine clearance, serum cystatin C and serum beta-trace protein. Iron status was evaluated by determination of serum iron, transferrin, transferrin saturation and serum ferritin. All determinations were performed using commercial reagents (Roche Diagnostics, Dade Behring). Serum prohepcidin was determined using an ELISA kit., Results: Serum prohepcidin was found to correlate with 51Cr-EDTA clearance (r = -0.44; p = 0.005), creatinine clearance, serum creatinine, beta-trace protein and cystatin C. No significant relationship was observed between serum prohepcidin concentrations and red cell count, hemoglobin concentration or hematocrit. No significant correlation was found in this population between prohepcidin concentrations and iron status., Conclusion: Increased serum prohepcidin concentrations were observed with declining kidney function. We observed no relationship between red cell indices or iron status and serum prohepcidin concentrations.

Carbohydrate-deficient transferrin (CDT) is currently considered to be the best available marker for the diagnosis of chronic alcoholism. A large variety of methods have been developed, demonstrating the need for standardisation. Commercially available anion-exchange chromatographic-based assays are easy to use and require no specialised, expensive instruments. However, these methods cannot identify genetic transferrin variants or the carbohydrate-deficient glycoprotein syndrome. In 1989, a capillary isoelectric focusing method was developed for quantitative measurement of CDT. Despite the optimal resolution, this method is not easily applied in a clinical routine environment due to the complexity of analysis. Capillary electrophoresis in a polymer network using coated capillaries allowed full resolution of the sialoforms of human transferrin. The drawbacks due to an expensive and time-consuming sample preparation were eliminated when a method in neat serum was developed. Capillary zone electrophoresis allowed full resolution of the transferrin isoforms with a high analytical performance in a short analysis time thanks to a strong electroosmotic flow. Genetic transferrin variants were easily detected, avoiding false-positive results. Also, using capillary zone electrophoresis, it was shown that CDT is a suitable marker of chronic alcohol abuse detection in transferrin CD (common/cathodal) variants.

Background: Creatine is widely used as an ergogenic substance among athletes. Safety of prolonged creatine intake has been questioned, based upon case reports and animal data. We investigated the effect of prolonged creatine ingestion on renal function in animals with normal kidney function or pre-existing kidney failure, respectively., Methods: Male Wistar rats were randomly allocated to four experimental groups: (i) sham-operated, control diet; (ii) sham-operated, creatine-supplemented diet (2% w/w (0.9+/-0.2 g creatine/kg body weight/day)); (iii) two-thirds nephrectomized, control diet; and (iv) two-thirds nephrectomized, creatine supplemented diet. Glomerular filtration rate was determined using inulin and creatinine clearance, together with albumin excretion, urea clearance, muscle and serum creatine and serum cystatin C concentrations., Results: In contrast to previous reports, no detrimental effects of creatine supplementation on the renal function indices were observed in two-thirds nephrectomized or sham-operated animals. No differences were observed in inulin (0.28+/-0.08 vs 0.25+/-0.08 ml/min/100 g; P=NS) or creatinine clearance rates. Serum cystatin C concentration, urinary protein excretion, and albumin and urea clearance were comparable between creatine-supplemented and control-diet fed animals in both sham-operated and two-thirds nephrectomized animals. Serum creatine and intramuscular total creatine concentrations were higher in creatine-supplemented groups (P<0.05)., Conclusions: Creatine supplementation at a dosage of 2% w/w for 4 weeks does not impair kidney function in animals with pre-existing renal failure or in control animals.

We discovered a hitherto undescribed insertion/deletion (I/D) polymorphism of 7 base pairs in intron 4 of the HP1 allele and intron 4 and 6 of the HP2 allele. Genotyping was performed in 311 Belgian subjects. The association between serum haptoglobin (Hp) and lipid concentrations and the Hp I/D genotypes was investigated. Genotype distribution in 103 Hp 1-1 phenotypes (50.5% DD, 39.8% DI, 9.7% II) and D allele frequency (0.70) were in close agreement with the Hardy-Weinberg equilibrium. No association between Hp concentrations and the Hp I/D genotypes could be found. Apolipoprotein (apo)A2, apoB and cholesterol concentrations were slightly lower in Hp II compared to DI and DD. Low-density lipoprotein (LDL)-cholesterol and ultrasensitive C-reactive protein (CRP) concentrations and the apoA1/apoA2 ratios differed significantly between Hp D/I genotypes. We added a further component to the molecular heterogeneity of Hp by the detection of an I/D polymorphism. Studies on the Hp I/D polymorphism in various populations open perspectives for further investigation of the distribution pattern of the human Hp gene. The association of the Hp I/D polymorphism with various lipid parameters might add a further component to the complex and multifaceted lipid metabolism.

Carbohydrate-deficient transferrin (CDT) is widely accepted as screening test for excessive alcohol consumption. However, results from subjects with transferrin variants must be interpreted with caution since chromatography-based methods may give false-positive results. Furthermore, due to the co-elution in HPLC or the co-migration in capillary zone electrophoresis (CZE) of the di- and trisialylated C transferrins with the tetrasialylated D peak, exact measurement of CDT is impossible in CD-variants. Therefore, in this study, we tried to offer a different solution, including only the asialo-D, asialo-C, monosialo-D, monosialo-C, disialo-D and trisialo-D transferrins in the CDT calculation and referring to a different cut-off value for CDT in transferrin CD-variants. Comparison of alcohol consumers with teetotalers demonstrated area under the receiver operating characteristic curve of 0.79 and 0.76 for carbohydrate-deficient transferrin, 0.71 and 0.71 for mean corpuscular volume and 0.51 and 0.68 for gamma-glutamyltransferase in 43 subjects with transferrin CD-variants and 225 subjects with CC-phenotypes, respectively. Since false-positive carbohydrate-deficient transferrin results due to a transferrin CD-variant have major social implications, capillary electrophoresis-based or similar methods (HPLC, FPLC) should be preferred in populations carrying a high D-allele frequency.

Background: Current methods for carbohydrate-deficient transferrin (CDT) often suffer from low precision, complexity, or risk of false positives attributable to genetic variants. In this study, a new capillary zone electrophoresis (CZE) method for CDT was developed., Methods: CZE was performed on a P/ACE 5000 using fused-silica capillaries [50 microm (i.d.) x 47 cm] and the CEOFIX CDT buffer system with addition of 50 microL of anti-C3c and 10 microL of anti-hemoglobin. Native sera were loaded by high-pressure injection for 3 s, separated at 28 kV over 12 min, and monitored at 214 nm., Results: CDT was completely resolved by differences in migration times (di-trisialotransferrin, 9.86 +/- 0.05 min; monosialotransferrin, 9.72 +/- 0.05 min; asialotransferrin, 9.52 +/- 0.04 min), with a CV of 0.15%. The number of theoretical plates was 312,000 +/- 21,000 for the mono- and 199 000 +/- 6500 for the di-trisialylated transferrin. Genetic CB and CD variants showed prominent peaks with migration times of 10.12 +/- 0.06 and 9.89 +/- 0.03 min, respectively, and the carbohydrate-deficient glycoprotein syndrome could be detected, excluding false-positive results. CZE results (as a percentage; y) correlated with the Axis %CDT TIA (x) values by Deming regression analysis: y = 1.92x - 7.29; r = 0.89. CDT values in 130 healthy nonalcoholics were determined. The 2.5th and 97.5th percentiles were 1.84% and 6.79%., Conclusions: CZE without sample pretreatment can determine CDT with good precision, allows detection of variants, and correlates with ion-exchange chromatography.

Background: Genetic variants of human transferrin (TF) have been described, but little is known about their functional differences. We studied iron status according to TF phenotype in a healthy Zimbabwean population and in subjects at risk of African iron overload., Methods: The study population consisted of 483 nondrinkers, 31 drinking spouse pairs, and 5 family pedigrees (n = 88) with index cases of iron overload. TF phenotypes were determined using starch gel electrophoresis. To evaluate iron status, serum iron, total iron-binding capacity (TIBC), ferritin, and soluble TF receptors were measured, and the percentage of saturation and the serum iron:TF ratio were calculated. The binding of the TF variants was studied by equilibrium dialysis., Results: The reference population was characterized by a high TF D allele frequency (0.050) and a complete absence of homozygous TF DD individuals. Similar allele frequencies were observed in subjects at risk of African iron overload. In the reference population, male TF CD heterozygotes had significantly lower (P <0.01) values for serum iron, TIBC, TF saturation, and serum iron:TF ratio than the TF CC homozygotes; in females, only TIBC was significantly different. Overall red blood cell indices did not differ according to TF phenotype. In the population at risk of African iron overload, only serum iron:TF ratio was consistently significantly lower in TF CD phenotypes (P <0.05). After equilibrium dialysis, the amount of iron bound by TF was significantly lower (P <0.01) in TF CD individuals., Conclusions: The present data demonstrate a functional difference between TF phenotypes in blacks.

A capillary zone electrophoresis method was developed for haptoglobin (Hp) phenotyping in hemoglobin (Hb) supplemented serum. The method allows a complete resolution of the major haptoglobin phenotypes Hp 1-1, Hp 2-1, and Hp 2-2 based on the difference in charge-to-mass ratio of their Hb-Hp complexes. Identification of these phenotypes was achieved by their significant differences in migration times and their marked difference in electrophoretic pattern. Our method showed full agreement with starch gel electrophoresis. Furthermore, following neuraminidase treatment of the serum, the Hp subtypes Hp1-1FF, FS and SS could be resolved, based on the same criteria as the phenotyping. The new electrophoretic method allowed typing of the rare phenotypes Hp 2-1 modified (Hp 2-1M) and Hp Johnson. The calculated hemoglobin binding capacity of serum correlates well with the nephelometrically determined haptoglobin concentration. The new method for typing haptoglobin gives prospectives for fast haptoglobin typing and Hp 1-1 subtyping.

The haptoglobin (Hp) 2-1 and 2-2 phenotypes have been shown to agglutinate Streptococcus pyogenes carrying the membrane antigen T4. In this study, the growth rate of two strains of Streptococcus pyogenes (T1 and T4) in human serum was compared among haptoglobin phenotypes in vitro. During incubation for 16 hours in serum of different haptoglobin types, only Hp 2-1 and Hp 2-2 sera showed an inhibitory effect on growth, Hp 2-2 being 1.85 times more potent than Hp 2-1. Growth of Streptococcus pyogenes T4 negatively correlated with the serum concentration of Hp 2-1 (r = -0.908) and Hp 2-2 (r = -0.953). Haptoglobin-depleted serum had no inhibitory effect on bacterial growth. Addition of haemoglobin and ferric citrate to the serum accelerated the growth of Streptococcus pyogenes T4 (P <0.05) but not in Hp 2-2 serum. Haptoglobin types 2-1 and 2-2 can be regarded as inhibitors of Streptococcus pyogenes growth in vitro. These data point towards a potential protective role of Hp 2-2 in Streptococcus pyogenes infection in vivo, independently of iron uptake.