GC–MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaffé, HPLC and enzymatic assays

In consideration of its relatively constant urinary excretion rate, creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous biomolecules. Assays based on the reaction of creatinine and picric acid first reported by Jaffé in 1886 still belong to the most frequently used laboratory approaches for creatinine measurement in urine. Further analytical methods for creatinine include HPLC–UV, GC–MS, and LC–MS and LC–MS/MS approaches. In the present article we report on the development, validation and biomedical application of a new GC–MS method for the reliable quantitative determination of creatinine in human urine, plasma and serum. This method is based on the derivatization of creatinine (d₀-Crea) and the internal standard [methyl-trideutero]creatinine (d₃-Crea) with pentafluorobenzyl (PFB) bromide in the biological sample directly or after dilution with phosphate buffered saline, extraction of the reaction products with toluene and quantification in 1-μl aliquots of the toluene extract by selected-ion monitoring of m/z 112 for d₀-Crea-PFB and m/z 115 for d₃-Crea-PFB in the electron-capture negative-ion chemical ionization mode. The limit of detection of the method is 100 amol of creatinine. In an inter-laboratory study on urine samples from 100 healthy subjects, the GC–MS method was used to test the reliability of currently used Jaffé, enzymatic and HPLC assays in clinical and occupational studies. The results of the inter-laboratory study indicate that all three tested methods allow for satisfactory quantification of creatinine in human urine. The GC–MS method is suitable for use as a reference method for urinary creatinine in humans. In serum, creatine was found to contribute to creatinine up to 20% when measured by the present GC–MS method. The application of the GC–MS method can be extended to other biological samples such as saliva.     

Automated measurement of urinary creatinine by multichannel kinetic spectrophotometry

Urinary creatinine analysis is required for clinical diagnosis, especially for evaluation of renal function. Creatinine adjustment is also widely used to estimate 24-h excretion from spot samples. Few convenient validated approaches are available for in-house creatinine measurement for small- to medium-scale studies. Here we apply the Jáffe reaction to creatinine determination with zone fluidic multichannel kinetic spectrophotometry. Diluted urine sample and reagent, alkaline picric acid, were mixed by a computer-programmed dispenser and rapidly delivered to a four-channel detection cell. The absorbance change was monitored by a flow-through light-emitting diode-photodiode-based detector. Validation results against high-performance liquid chromatography-ultraviolet (HPLC-UV)/mass spectrometry (MS) are presented. Responses for 10-fold diluted samples were linear within clinically relevant ranges (0-250 mg/L after dilution). The system can analyze 70 samples per hour with a limit of detection of 0.76 mg/L. The relative standard deviation was 1.29% at 100 mg/L creatinine (n=225). Correlation with the HPLC (UV quantitation/MS confirmation) system was excellent (linear, r2=0.9906). The developed system allows rapid, simple, cost-effective, and robust creatinine analysis and is suitable for the analysis of large numbers of urine samples.     

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography-mass spectrometry

4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography-mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 microl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28-2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.     

A New Enzymatic Method for the Measurement of Creatinine Involving a Novel ATP-dependent Enzyme,N-Methylhydantoin Amidohydrolase

A new enzymatic method for the measurement of serum and urine creatinine is described. The method is based on a novel microbial creatinine degradation pathway via N-methylhydantoin [Shimizu et al., Clin. Chim. Acta, 185, 241–252 (1989)]. By using two novel enzymes, N-methylhydantoin amidohydrolase and N-carbamoylsarcosine amidohydrolase, as key enzymes, coupled with a colorimetric procedure for hydrogen peroxide detection, the creatinine level can be measured. The results obtained for human serum and urine show good correlation with those obtained by a standard chemical method based on the Jaffe reaction. The new method is simple and specific, and shows excellent sensitivity and reliability.     

Autoanalytical procedure for the determination of allantoin and allantoic Acid in soybean tissue

Ureide analyses of soybean (Glycine max L.) tissues were accomplished with a modified and simplified automated analysis used to determine allantoin concentration in rat urine. The length of the circuit and flow rates of the solutions were reduced, and NaOH was used for color development at room temperature. Keto-acids did not significantly interfere with the determinations of ureides except for glyoxylic acid in extracts of fresh soybean tissue. The interference caused by glyoxylic acid was avoided by adding phenylhydrazine HCl to the solution of NaOH used for alkaline hydrolysis of allantoin.     

Assessment of serum beta-trace protein (BTP) measurement in the prediction of glomerular filtration rate. Comparison with serum cystatin C

Serum BTP measurement is sometimes believed to be an alternative marker of glomerular filtration rate (GFR) assessment. The aim of the present work was to investigate the correlation between creatinine, cystatin C, and BTP values in sera and to compare the diagnostic efficacy for serum BTP and cystatin C with the glomerular filtration rate estimate. 25 individuals were tested. GFR was estimated from creatinine clearance, serum cystatin C and BTP and urine alpha-1 microglobulin, albumin, GMT and creatinine were measured. BTP values correlated with cystatin C (r = 0.75; p < 0.01), creatinine (r = 0.73, p < 0.01), GFR (r = -0.46; p = 0.02), urine alpha-1-microglobulin (r = 0.66; p < 0.01). The diagnostic efficacy of BTP for reduced GFR was insufficient and the calculation of GFR with BTP was not included in the regression model.     

Über die Gewöhnung von Ziegen an eiskaltes Tränkwasser

90 urine samples obtained in three lamb trials and one experiment using adult wethers were analyzed for their contents of orotic acid and creatinine. The average daily excretion of orotic acid accounted for 0.5 mg to 1.5 mg (35 μg to 130 μg/W^0.75) with a high individual variation. Correlation coefficients between orotic acid and other urinary constituents were low indicating an entirely different response to metabolic variations. There was only a weak relationship to live weight, protein retention and rumen fluid traits. Defaunation reduced the orotic acid excretion (significant in the adult wethers) whereas the addition of rumen‐protected lysine as well as the use of different dietary carbohydrate sources were without effect. The urinary excretion of creatinine increased with live weight and age from 0.4 g/d in the 20 kg lambs to 1.7 g/d in the adult 53 kg wethers. The correlations with live weight were close whereas the apparently negative correlation with protein retention was not real as could be evaluated by calculation of the partial correlations. There was a close correlation of creatinine with total N, urea and allantoin. Neither defaunation nor rumen‐protected lysine and the kind of carbohydrate source had significant effects on creatinine. The use of orotic acid and creatinine as indicators of metabolic disorders were discussed. Easy application in practical diagnosis without quantitative urine collection might be possible by the determination of orotic acid in the milk of cows and of the creatinine/N ratio in urine.     

Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography

The creatinine levels of blood and urine from humans, rats, and mice were measured by high-performance liquid chromatography. These were compared to the alkaline picrate analysis of creatinine performed by standard colorimetric, kinetic, and AutoAnalyzer techniques. For human serum and urine the values obtained using the HPLC technique gave good agreement with four out of five alkaline picrate techniques. For black or white mice, the serum creatinine concentration was 8.7 +/- 0.4 microM by HPLC but 44.9 +/- 1.9 microM by the lowest alkaline picrate method. Mouse urine creatinine concentrations were 3.24 +/- 0.19 mM by HPLC and 4.59 +/- 0.39 mM by the nearest alkaline picrate method. Rat serum creatinine concentrations analyzed by HPLC were about half the values obtained by AutoAnalyzer. Mouse and rat samples seemed to have substances which gave nonspecific color and thus interfered with the analysis of creatinine by the alkaline picrate methods. While the alkaline picrate analysis of creatinine was adequate for human samples, it was necessary to use HPLC to accurately measure rodent creatinine. The fractional excretion of creatinine was determined by measuring creatinine in mouse urine and plasma by both the kinetic and HPLC methods and comparing these values to urine and plasma inulin. Using the kinetic method, creatinine was cleared at 43 +/- 3% of the rate of inulin. Using the HPLC method, creatinine was cleared at 170 +/- 11% of the rate of inulin.     

Specific gravity as an alternative to creatinine for estimating urine concentration in captive and wild chimpanzee (Pan troglodytes) Samples

The measurement of hormones in urine has become a widely used technique in primatology. Because urine concentration varies according to fluid intake, concentration must be measured in each sample collected, and hormone values are always expressed per unit of concentration. Traditionally, creatinine has been used as a concentration index, but some studies in humans have shown that creatinine varies among populations and even within and between individuals within a population, and that it begins to degrade after just one freeze-thaw cycle. In addition, creatinine measurement is relatively time-consuming and expensive and creates hazardous waste. In this study, we tested the hypothesis that specific gravity, or the ratio of the density of a sample to that of water, is highly correlated with creatinine measurement in urine samples collected from captive chimpanzees at the New Iberia Research Center in Louisiana and wild chimpanzees at the Ngogo study site in the Kibale National Park, Uganda. We found that specific gravity and creatinine were highly correlated in both captive (N=124) and wild (N=13) chimpanzee samples, and that specific gravity measurement was robust to actual and simulated transport conditions and repeated freeze-thaw cycles. We recommend that researchers consider specific gravity measurement as a preferable alternative to creatinine measurement in their studies of primate endocrinology.     

Quantification of urinary 5-aminolevulinic acid by gas chromatography-mass spectrometry

In this report, we describe a method for the specific quantification of urinary 5-aminolevulinic acid. It is based on gas chromatographic mass spectrometric measurement of the trimethylsilyl ester of the ethylacetoacetate pyrrole derivative of 5-aminolevulinic acid. Selected ion monitoring (SIM) was used for quantification using 3-hydroxymyristic acid as an internal standard. The detection limit of this method was 0.1 nmol/ml. This method was applied to the determination of urinary 5-aminolevulinic acid from a tyrosinemia type I patient and normal subjects, and 21.4 mmol/mol creatinine and 0.54+/- 0.49 mmol/mol creatinine (n = 7), respectively, were detected. Less than 0.2 ml urine was sufficient for the determination of 5-aminolevulinic acid in healthy subjects.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

Separation and quantitation of oxypurines by isocratic high-pressure liquid chromatography: application to xanthinuria and the Lesch-Nyhan syndrome

An isocratic HPLC technique has been developed for the separation and measurement of urine and plasma oxypurines in a patient with xanthinuria. The case history and laboratory data are presented. Xanthine excretion was 172 mg/g creatinine and hypoxanthine was 45 mg/g creatinine. Uric acid was too small to be measured but uricase determination showed only 3 mg/24 hr. Serum oxypurine analysis showed hypoxanthine 0.87 mg/dl and xanthine 0.35 mg/dl. Uric acid was not seen in this patient's serum but could be readily measured in normal control subjects. The technique can also be used to separate nucleotides from purine bases, and we have demonstrated its application to the measurement of erythrocyte hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase in a kindred associated with the Lesch-Nyhan syndrome.     

6周大强度训练对大鼠肾功能的影响及其机制

目的：研究6周大强度训练对大鼠肾功能影响及运动性蛋白尿的机制。方法：6周龄SD雄性大鼠36只随机分为：安静对照组（C组,n=12）和大强度训练组（M组,n=24）。大鼠适应性饲养4 d后,C组不进行任何运动,M组采用6周递增负荷游泳训练。每周训练6 d,每天1次。第4周起开始负重（体重的1%）并逐渐递增（体重的6%）。各组大鼠末次训练结束后30 min取单次尿,24 h后腹主静脉取血,取双侧肾脏待测。HE染色观察肾脏肾小球结构,酶联免疫吸附法测定尿总蛋白、尿微量白蛋白、中性粒细胞明胶酶相关脂质运载蛋白,碱性苦味酸法测试尿肌酐,比色法测定血清肌酐、尿素氮,蛋白质免疫印迹法检测肾组织Nephrin蛋白表达量,放射免疫法测试血清睾酮、皮质酮和肾组织及血液中肾素-血管紧张素系统相关指标变化。结果：与C组比较,M组血清睾酮/皮质酮显著降低（P<0.01）;尿液蛋白总量、微量白蛋白、微量白蛋白与肌酐比值、中性粒细胞明胶酶相关脂质运载蛋白、血清尿素氮、肌酐均显著升高（P<0.01）;肾小球结构明显改变,且Paller评分明显增高（P<0.01）;肾组织Nephrin蛋白表达量明显降低（P<0.01）;肾脏内部与循环血液中肾素活性、血管紧张素Ⅱ显著增高（P<0.01）。结论：6周大强度训练对大鼠肾功能影响及运动性蛋白尿的机制可能为过度训练诱导肾脏内部及循环系统中肾素-血管紧张素系统持续兴奋,并下调Nephrin蛋白的表达,进而导致肾脏结构与功能异常,出现蛋白尿。     

Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.     

Rapid method for the determination of lysine in cereal grains without hydrolysis.

A rapid spectrophotometric method for lysine determination in cereal grains is reported. The reagent is sodium dinitrobenzene sulfonate (DNBS). The absorbance of the acid solution of the DNBS derivative is read at 385 nm. Under prescribed conditions, the reagent is sensitive and specific for free or protein-bound lysine. The conditions: For rice, the reaction medium is a urea-phosphate-HgCl₂ solution (pH 10.5); reaction at 60°C for 1 hr. For other grains, the reaction medium is a urea-phosphate-phenylmercuric chloride (PMC) solution (pH 12.3); reaction at 40°C for 1 hr. Interferences are eliminated by ether extraction after color development and acidification and addition of formic acid and the sulfhydryl masking agents, HgCl₂ or phenylmercuric chloride (PMC). NaOH extracts of cereal proteins are used for analysis. Values are in agreement with those of the ion-exchange amino acid analyzer.     

A rapid, sensitive, and specific method for the determination of protein in dilute solution

A protein assay is described in which the sample is precipitated with trichloroacetic acid in the presence of sodium dodecylsulfate, filtered off on a Millipore membrane and stained with Amidoschwarz 10B. The proteindye complex is eluted, and its absorbance determined at 630 nm. This assay is very reproducible, insensitive to variations in assay conditions, and linear from 3 to 30 μg of protein. It can be used on samples with a concentration as low as 0.75 μg/ml. There is no interference by commonly used reagents such as Tris, thiol reagents, EDTA, urea, sucrose, and many others. The color yield for a variety of proteins was determined and found to lie within ±15% of the value for bovine serum albumin which was used as standard. Of the proteins tested only insulin, which due to its low molecular weight was incompletely retained on the membrane in the filtration step, gave a low color yield, 50% of the standard.     

The Plasma Phenolsulfonphthalein Index (PSPI) of Renal Function: II. Correlation with Other Parameters of Renal Function and Indications for Use

The plasma phenolsulfonphthalein index (PSPI) was determined in 175 subjects and was compared with levels of blood urea nitrogen (BUN) and serum creatinine, 15-minute urinary excretion of PSP, and clearance of creatinine, PAH and inulin. Statistical analyses indicate that the PSPI measures the same function as the 15-minute urine test when PSP is administered in a dosage of 1 mg./kg. body weight; and that, although the index cannot be used as a precise substitute for PAH clearance, it is equally repeatable and no greater error is associated with its measurement. The PSPI has been found of most value when complete, accurately timed urine collections are unobtainable because of urological abnormality or inability of the patient to co-operate.     

8-Oxo-2'-deoxyguanosine as a biomarker of tobacco-smoking-induced oxidative stress

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) is a useful biomarker of oxidative stress. However, its analysis can be challenging because 8-oxo-dGuo must be quantified in the presence of dGuo, without artifactual conversion to 8-oxo-dGuo. Urine is the ideal biological fluid for population studies, because it can be obtained noninvasively and it is less likely that artifactual oxidation of dGuo can occur because of the relatively low amounts that are present compared with hydrolyzed DNA. Stable isotope dilution liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM/MS) with 8-oxo-[(15)N(5)]dGuo as internal standard provided the highest possible specificity for 8-oxo-dGuo analysis. Furthermore, artifact formation was determined by addition of [(13)C(10)(15)N(5)]dGuo and monitoring of its conversion to 8-oxo-[(13)C(10)(15)N(5)]dGuo during the analytical procedure. 8-Oxo-dGuo concentrations were normalized for interindividual differences in urine flow by analysis of creatinine using stable isotope dilution LC-SRM/MS. A significant increase in urinary 8-oxo-dGuo was observed in tobacco smokers compared with nonsmokers either using simple urinary concentrations or after normalization for creatinine excretion. The mean levels of 8-oxo-dGuo were 1.65ng/ml and the levels normalized to creatinine were 1.72μg/g creatinine. Therefore, stable isotope dilution LC-SRM/MS analysis of urinary 8-oxo-dGuo complements urinary isoprostane (isoP) analysis for assessing tobacco-smoking-induced oxidative stress. This method will be particularly useful for studies that employ polyunsaturated fatty acids, in which a reduction in arachidonic acid precursor could confound isoP measurements.     

Detection of Novel Visible-Light Region Absorbance Peaks in the Urine after Alkalization in Patients with Alkaptonuria

Background

Alkaptonuria, caused by a deficiency of homogentisate 1,2-dioxygenase, results in the accumulation of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) in the urine. Alkaptonuria is suspected when the urine changes color after it is left to stand at room temperature for several hours to days; oxidation of homogentisic acid to benzoquinone acetic acid underlies this color change, which is accelerated by the addition of alkali. In an attempt to develop a facile screening test for alkaptonuria, we added alkali to urine samples obtained from patients with alkaptonuria and measured the absorbance spectra in the visible light region.

Methods

We evaluated the characteristics of the absorption spectra of urine samples obtained from patients with alkaptonuria (n = 2) and compared them with those of urine specimens obtained from healthy volunteers (n = 5) and patients with phenylketonuria (n = 3), and also of synthetic homogentisic acid solution after alkalization. Alkalization of the urine samples and HGA solution was carried out by the addition of NaOH, KOH or NH4OH. The sample solutions were incubated at room temperature for 1 min, followed by measurement of the absorption spectra.

Results

Addition of alkali to alkaptonuric urine yielded characteristic absorption peaks at 406 nm and 430 nm; an identical result was obtained from HGA solution after alkalization. The absorbance values at both 406 nm and 430 nm increased in a time-dependent manner. In addition, the absorbance values at these peaks were greater in strongly alkaline samples (NaOH- KOH-added) as compared with those in weakly alkaline samples (NH4OH-added). In addition, the peaks disappeared following the addition of ascorbic acid to the samples.

Conclusions

We found two characteristic peaks at 406 nm and 430 nm in both alkaptonuric urine and HGA solution after alkalization. This new quick and easy method may pave the way for the development of an easy method for the diagnosis of alkaptonuria.     

Urinary metanephrines by liquid chromatography tandem mass spectrometry: using multiple quantification methods to minimize interferences in a high throughput method

Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC-MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L. The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC-MS method. Injection-to-injection time was 6 min. The validated LC-MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well.