Modulation of Gold Nanorod Growth via the Proteolysis of Dithiol Peptides for Enzymatic Biomarker Detection.

Gold nanorods possess optical properties that are tunable and highly sensitive to variations in their aspect ratio (length/width). Therefore, the development of a sensing platform where the gold nanorod morphology (i.e., aspect ratio) is modulated in response to an analyte holds promise in achieving ultralow detection limits. Here, we use a dithiol peptide as an enzyme substrate during nanorod growth. The sensing mechanism is enabled by the substrate design, where the dithiol peptide contains an enzyme cleavage site in-between cysteine amino acids. When cleaved, the peptide dramatically impacts gold nanorod growth and the resulting optical properties. We demonstrate that the optical response can be correlated with enzyme concentration and achieve a 45 pM limit of detection. Furthermore, we extend this sensing platform to colorimetrically detect tumor-associated inhibitors in a biologically relevant medium. Overall, these results present a subnanomolar method to detect proteases that are critical biomarkers found in cancers, infectious diseases, and inflammatory disorders.

Urinary aprotinin as a predictor of acute kidney injury after cardiac surgery in children receiving aprotinin therapy.

Proteomic analysis has revealed potential early biomarkers of acute kidney injury (AKI) in children undergoing cardiopulmonary bypass (CPB), the most prominent one with a mass-to-charge ratio of 6.4 kDa. The objective of this study was to identify this protein and test its utility as a biomarker of AKI. Trypsin-digested protein bands were analyzed by tandem mass spectrometry (MS/MS) to identify the protein in urine samples. Surface-enhanced laser desorption/ionization time-of-flight analysis and a functional activity assay were performed to quantify urinary levels in a pilot study of 106 pediatric patients undergoing CPB. The protein was identified as aprotinin. Urinary aprotinin levels 2 h after initiation of CPB were predictive of AKI (for functional assay: 92% sensitivity, 96% specificity, area under the curve of 0.98). By multivariate analysis, the urinary aprotinin level 2 h after CPB was an independent predictor of AKI (beta = 0.001, P < 0.0001). The 2 h urinary aprotinin level correlated with serum creatinine, duration of AKI, and length of hospital stay. We concluded that urinary aprotinin levels 2 h after initiation of CPB predict the development of AKI and adverse clinical outcomes.

Pharmacokinetics and normal scintigraphic appearance of 99mTc aprotinin.

OBJECTIVE: To confirm the pharmacokinetics and biodistribution of 99mTc aprotinin in normal volunteers and to determine the optimum time for scanning post-injection, prior to further investigations of 99mTc aprotinin as an imaging agent for amyloidosis. METHODS: Five patients (three men and two women, average age 49 years, age range 38-66 years) without a history of amyloidosis or any of the associated diseases, were included in this prospective study. Blood and urine were collected and images were performed of the whole body and wrists. CONCLUSIONS: Normal biodistribution of 99mTc aprotinin includes early cardiac and lung activity in the blood pool phase with subsequent hepatic activity and renal excretion with variable splenic activity. There is variable bowel uptake on later images. The best quality images were obtained 90 min post-intravenous administration, and this is likely to be the optimum time for clinical imaging.

Tubular peptide hypermetabolism and urinary ammonia in chronic renal failure in man: a maladaptive response?

Excessive renal tubular peptide uptake and degradation reflecting hypercatabolism may be a maladaptive response in chronic renal failure (CRF). It may also offer an explanation for the increased ammoniagenesis, per surviving nephron, observed in CRF but as yet unexplained. Neither has been explored in man. We have shown in patients with normal renal function and heavy (>5.0 g/24 h) proteinuria that tubular catabolism of a technetium-labelled peptide marker, aprotinin, and urinary ammonia were increased compared to others with less proteinuria. We now measure tubular kinetics of aprotinin and urinary ammonia in 16 CRF patients with variable proteinuria. Metabolism and turnover of aprotinin and ammonia excretion were increased, corrected for glomerular filtration rate, to levels found in patients with normal function and heavy proteinuria.

Aprotinin prevents the development of the trauma-induced multiple organ failure in a chronic sheep model.

Trauma-induced multiple organ failure in sheep was prevented by aprotinin therapy. Multiple organ failure was induced in 16 female merino sheep by initial haemorrhagic shock and intramedullary femoral nailing (day 0), and 12 hourly injections of 0.75 micrograms/kg Escherichia coli endotoxin +0.7 ml/kg zymosan-activated plasma (days 1-5). In addition, the aprotinin group (n = 6) received simultaneous injections of 5 mg/kg (35 695 KIU/kg) aprotinin, whereas ten animals did not receive aprotinin and served as the control group (n = 10). Organ functions were monitored for a total of 11 days by measuring haemodynamic, cardio-respiratory and biochemical quantities of blood, urine and epithelial lining fluid. During the subsequent eleven day period, aprotinin induced a significant (p < 0.05) reduction of the pathological changes (development of multiple organ failure) seen in the control group. Thus, aprotinin prevented an alteration of cardiac function (cardiac index for control/aprotinin groups at day 1: 6.5/6.2, and at day 10: 10.47/7.0 1/min x m2), an impairment of lung function (mean pulmonary arterial pressure at day 1: 2.26/1.86, and at day 10: 3.83/2.13 kPa; epithelial lining fluid/plasma ratio of albumin concentrations as a direct marker of lung capillary permeability damage at day 0: 0.18/0.16, and at day 10: 0.45/0.15), a deterioration of liver function (plasma sorbitol dehydrogenase at day 0: 7.9/7.6, and at day 10: 29.6/7.4 U/1), but not of renal function (creatinine clearance at day 1: 91.4/66.1, and at day 10: 53.1/59.2 ml/min). Urinary aprotinin excretion increased up to day 3, then decreased rapidly despite further aprotinin administration. As a non-specific marker of cell damage, plasma lactate dehydrogenase indicated an aprotinin-induced organ protection (day 0: 501/409, and at day 10: 719/329 U/1). The neutrophil count and the measured chemiluminescence of neutrophils from the blood and epithelial lining fluid showed that aprotinin reduced the in vivo neutrophil activation, the alveolar neutrophil invasion, the production of inflammatory mediators, and the production of reactive oxygen metabolites during the passage of the capillary-interstitial-alveolar space by neutrophils.

Glomerular filtration and tubular absorption of the basic polypeptide aprotinin.

The basic polypeptide aprotinin (Ap), mol. wt 6500, pI 10.5, is filtered in the glomeruli, virtually completely taken up by the proximal tubular cells and retained there for many hours. This process was studied in rats by determining the renal plasma clearance (CAp) as the amount of [125I]Ap accumulated in the kidney plus that excreted in the urine per unit of time divided by the integrated plasma concentration. In periods lasting 4-20 min after i.v. bolus injection or infusion to constant plasma concentration, CAp was 65% of glomerular filtration rate (GFR) estimated as kidney plus urinary clearance of [51Cr]EDTA (Ccr-EDTA). Less than 0.8% of the filtered Ap appeared in the urine. CAp varied inversely with plasma protein concentration in mg ml-1: CAp/Ccr-EDTA = 0.98-0.0058 x Ppr, corresponding to a glomerular Gibbs-Donnan distribution for a net molecular charge of +6, in agreement with the amino acid composition of Ap. CAp (kidney + urinary) was not altered by inhibiting tubular uptake of [125I]Ap by maleate or by exceeding the uptake capacity with large doses of unlabelled Ap. Neutralized Ap (malonylated) did not accumulate in the kidney, but showed a urinary clearance indistinguishable from that of [51Cr]EDTA. Both CAp and Ccr-EDTA were reduced to 0.04 ml min-1 when glomerular filtration pressure was lowered by ureteral stasis and increased Ppr (80-90 mg ml-1). These findings indicate: (1) no steric or charge restriction to filtration of Ap in the glomerular membrane, (2) the Gibbs-Donnan equilibrium should be considered when estimating glomerular sieving of charged polypeptides in intact animals (3) charge dependent tubular uptake, (4) little or no transtubular transport of intact Ap, (5) no appreciable tubular uptake of Ap from the peritubular side and (6) local renal accumulation of Ap in a period of up to 20 min may be used to estimate local glomerular filtration and/or local proximal tubular reabsorption rates. Model analysis based on the appearance of 125I in plasma, the time course of renal Ap content, and literature data on subcellular Ap distribution are consistent with two populations of endosomes, transporting Ap at widely different rates from the proximal tubular brush border to the lysosomes where breakdown occurs at a high rate.

Repeatable measurement of local and zonal GFR in the rat kidney with aprotinin.

The basic polypeptide aprotinin (Ap), mol. wt 6513, is freely filtered in glomeruli and completely reabsorbed by the proximal tubules. Cellular processing is slow with return to plasma of breakdown products beginning after 20-30 min. When corrected for Gibbs-Donnan distribution of Ap between glomerular filtrate and plasma (i.e. 0.65 at a plasma protein concentration of 50 mg ml-1), the renal clearance of [125I]Ap, estimated as the ratio of kidney uptake and integrated non-protein bound plasma 125I concentration, equals that of [51Cr]EDTA (urine + kidney content). Zonal GFR per gram tissue was obtained from uptake in three to six samples from outer and inner cortex (OC, IC) and the cortico-medullary border zone, 5-30 min after i.v. injection in rats. Control GFR in OC was 2.05 (SD 0.39) ml g-1 min-1 and the IC/OC ratio 0.66 (SD 0.14). Repeated local clearances (CI and CII) were obtained by injecting a second tracer (i.e. [131I]Ap) 15 min after the first injection (i.e. [125I]Ap), which by then had a low plasma concentration. The kidneys were removed at 30 min, frozen and dissected. During control conditions CII/CI averaged 1.06 in OC and IC, and the coefficient of variation (CV) between CII/CI ratios of individual tissue samples was 2% in both zones. Lowering left renal arterial pressure before the second injection reduced GFR proportionally in both zones (34 and 37%) with a CV of intersample CII/CI ratios of 5%. We conclude that the method allows precise and repeatable measurements of local and zonal GFR.

A new method to measure renal tubular degradation of small filtered proteins in man using radiolabelled aprotinin (Trasylol).

1. A method has been developed to measure the renal tubular degradation of small filtered proteins in man using radiolabelled aprotinin (Trasylol), a 6500 Da cationic polypeptide. 2. Aprotinin (0.5 or 5.0 mg) was labelled with either 99mTc (40 MBq) or 131I (1 MBq) and injected intravenously in 19 renal patients (10 with normal renal function and nine on haemodialysis). Activity in plasma and urine was measured over 48 h, and chromatography with Sephadex-G-25-M was used to separate labelled aprotinin from free 99mTcO4- or 131I-. Renal uptake was measured for 99mTc-labelled aprotinin only. 3. The volumes of distribution were similar in all patients: 18.2 +/- 0.4 litres in those with normal renal function and 20.2 +/- 0.1 litres in the others. Chromatography showed all plasma activity as undegraded aprotinin and urine activity only as the free labels (99mTcO4- or 131I-). 4. In patients with normal renal function, activity in the kidneys rose rapidly to 24.2 +/- 2.8% of dose after 90 min and to 42.2 +/- 3.4% of dose after 24 h. In the dialysis patients, activity over the kidneys was only 2.7 +/- 0.8% of dose at 24 h. Extra-renal uptake was insignificant in all patients with normal kidney function. 5. Both 99mTcO4- and 131I- appeared in the urine promptly after injection, and the rates of excretion of the two isotopes were similar, varying little over 24 h (1.8 +/- 0.04% of dose/h and 1.7 +/- 0.04% of dose/h for 99mTc and 131I, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lysine infusion on the renal metabolism of aprotinin (Trasylol) in man.

1. Aprotinin (Trasylol) is a cationic 6500 Da polypeptide that inhibits proteolytic enzymes, and when labelled with 99mTc it is a reproducible marker for the renal tubular turnover of small filtered proteins in man. Lysine potently inhibits tubular peptide uptake, and may thus depress the uptake and metabolism of aprotinin. This was investigated in 14 glomerulonephritic patients with normal renal function and variable proteinuria and in one healthy subject. 2. 99mTc-labelled aprotinin was given intravenously alone, and again 3 days later, immediately after the intravenous administration of 3-6 g of lysine, followed by an infusion over 1 h of 0.3-1.9 g of lysine/kg in individual patients. Activity over kidneys and in urine was measured over 24 h and chromatography was used to separate the undegraded peptide from free isotope. 3. At the low dosage of lysine (< 0.8 g/kg) given to six patients, kidney activity (representing tubular uptake) was unchanged, but early urine samples contained some undegraded aprotinin. Urinary excretion of free isotope, representing tubular metabolism, fell from 1.6 +/- 0.2% of dose/h with no lysine to 0.9 +/- 0.1% of dose/h in the 24 h after lysine, suggesting suppression of tubular aprotinin degradation. Corrected fractional degradation was calculated from the mean urinary excretion of free isotope over a given interval, determined by chromatography, divided by the mean cumulative kidney counts over this same interval, and this also fell after lysine from 0.06 +/- 0.006 to 0.03 +/- 0.006 h-1 (P < 0.005) between 3.75 and 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal effects on kallikrein, esterase A2, and their inhibitors in rat urine.

The effects of various hormones on the urinary excretion of kallikrein and esterase A2 were studied in rats. Chronic treatment with antidiuretic hormone had no effect on the excretion of either enzyme. Deoxycorticosterone treatment or a low sodium diet stimulated urinary kallikrein excretion (as is well known), but had no effect on urinary esterase A2. Dexamethasone markedly suppressed the excretion of both kallikrein and esterase A2 and increased the excretion of proteins (inhibitors) that bind to each of these enzymes. It is likely that the suppressive effect of glucocorticoid on urinary kallikrein and esterase A2 activities is due to the increase in urinary binding proteins. There is also a strong correlation between the excretion of kallikrein and esterase A2 in normal untreated rats. Such a correlation might arise from a common effect of glucocorticoid-influenced inhibitors on both enzymes.

Components of the kallikrein-kinin system in urine.

The excretion of kallikrein in urine varies, but the pathophysiologic implications are not clear. To help clarify the role of the urinary kallikrein-kinin system, we have begun to define components of the system as they occur in urine. To minimize artifacts which may arise through extensive purification procedures, we studied urinary protein concentrates prepared by ultrafiltration. The concentrates were separated by chromatography on Sephacryl. Urine contains abundant kininase activity, but in strongly inhibited forms. Kininase II is separable into at least two forms. Another major kininase can hydrolyze benzoyl-Pro-Phe-Arg and is inhibited by arginine but not by BPP9a or SQ 14,225. Its molecular weight is approximately 63,000. A third kininase, not inhibited by BPP9a, is excluded from Sephacryl. Human urine appears to contain only one kallikrein-like enzyme (MW 45,000). In addition, urine contains a protein (MW approximately 80,000) which reacts with trypsin to release bradykinin and which inhibits the hydrolysis of Pro-Phe-Arg-[3H]anilide by urinary kallikrein. Thus, in addition to kallikrein and kinins, urine contains kininogen and at least three kininase enzymes. Urinary ultrafiltrate contains an inhibitory substance (approximately MW 400).