ASSESSMENT OF RENAL MALFUNCTION BY ESR SPECTROSCOPY

Abstract: A process for detecting and/or monitoring loss or impairment of renal function comprises reacting a clinical sample from a patient with suspected or confirmed loss or impairment of renal function with a compound capable of interacting with a generated free radical species to form a stable radical adduct and detecting the presence of such adduct by electron spin resonance spectroscopy. For this purpose an ESR spectrometer is provided, adapted to operate over a restricted magnetic field width around the center field of the signal being detected.

Description: Assessment of renal malfunction by ESR spectroscopy. This invention relates to an assay for use in detecting and/or monitoring loss of renal function and is particularly 5 concerned with the monitoring of adequancy of dialysis in patients with impaired or no renal function. Malfunction of the kidneys is recognised as a very serious disorder, whether as acute kidney failure in formerly normal patients or as chronic renal failure in patients requiring a 10 continuous programme of dialysis in order to allow the body to continue to function satisfactorily. While some aspects of the chemistry associated with renal failure are known, the retention of certain products of normal biochemistry such as creatinine lead to the accepted methods of monitoring renal function. Serum 15 levels of creatinine depend upon its rate of production, and secretion, which occurs by filtration through the glo eruli and on renal tubular function. However, because creatinine secretion depends on glomerular filtration, and levels only rise when the glomerular filtration 20 rate has fallen by over 60% of normal, it is a poor measure of renal function and, in any case, only gives information on the single aspect of glomerular. filtration capacity. As a result, in renal impairment or failure, it only falls once glomerular filtration is restored and gives no information as to the 25 metabolic state of the kidney. Creatinine, βe_r ιe_, is not a toxic species, and there is considerable interest in what may be the biochemical products retained which cause the 'uraemic syndrome'. There is therefore a need for an improved method of more accurately monitoring loss or impairment of renal function, 30 and in particular for monitoring the efficiency of dialysis necessary for patients with impaired or no natural renal function. * It has now been found that an oxidant species characteristic of renal failure can be detected by electron spin resonance (esr) spectroscopy. Esr spectroscopy is a known spectroscopic 35 technique which has been used for many years as a research tool. The technique relies on trapping a generated free radical in the reaction being studied using a non-radical "spin trap" substance which interacts with the free radical species to give a stable adduct with a characteristic esr-spectrum. The technique has been used in some biological systems employing 3,5-dibromo-4- nitroso-benzene sulphonate (DBNBS) as spin trap. Using a superoxide generating system (xaπthine/xaπthine oxidase), a stable three line spectrum was produced, interpreted initially as a DBNBS-0 2 ~ -adduct (Ozawa and Hanaki , Biochem Biophys. Res. Commun. J3£, 1986, 657-664). The supernatant from Inflamed human synovia exposed to a hypoxia/normoxia cycle also caused a three line spectrum with DBNBS, the amplitude of which was reduced by either SOD or allopurinol (Allen, Slake £i al, Lancet, 1989 II, 282-283). The spectra were thus assigned to a superoxide spin adduct. Later work suggested the presence of contaminating peroxidases in the xanthine oxidase or SOD preparations used (Nazhat, Yang si il, Biochem. Biophys. Res. Commun., 16J>. 1990, 807-812). Therefore the three line spectrum was attributed to a 1-electron peroxidatic oxidation of DBNBS by an intermediate formed from a catalase or peroxidase with hydrogen peroxide (formed from the dismutation of superoxide), or from another peroxide in the system. It is against this background that it has now surprisingly been found that clinical samples from patients suffering renal failure contain a detectable free radical species, believed to be an oxidant species unaffected by superoxide dismutase and gamma-radiation, which free radical species appears to be unique to conditions associated with loss or impairment of renal function. Furthermore, it has been found that, as normal kidney function is restored (for example by dialysis) the concentration of the free radical species falls steadily- According to the present invention there is provided a process for detecting and/or monitoring loss or impairment of renal function comprising reacting a clinical sample from a patient with suspected or confirmed loss or impairment of renal function with a compound capable of interacting with a generated * free radical species to form a stable radical adduct and detecting the presence of such adduct by electron spin resonance % spectroscopy. 5 Preferably the compound capable of interacting with the generated radical species is a nitroso type compound, such as 3,5-dibromo-4-nitroso-benzene sulphonate (DBNBS) which is already known as a "spin trap" for use in esr spectroscopy and is commercially available. However, it is envisaged that other 10 nitroso compounds may be suitable as spin traps. The invention therefore further includes the use of DBNBS, and related nitroso type spin traps, for use in the detection of free radical species in clinical samples from patients with suspected or confirmed renal malfunction. 15 The clinical sample may be whole blood, or any fraction thereof, e.g. blood plasma, or alternatively a dialysate sample if the patient is undergoing renal or peritoneal dialysis, or a urine sample, for example of a patient with impaired kidney function. It will be appreciated that the process of the 20 invention is equally applicable to dialysis patients with impaired renal function or with total loss of natural renal function. Blood for analysis may be taken into a syringe as part of normal biochemical analysis. As the signal being detected is 25 present in whole blood, no separation of plasma or serum is necessary and the activity appears unaffected by anticoagulant. However separation of plasma or serum may be more convenient. Alternately, blood may be drawn directly into a vacutainer containing DBNBS, so that a known volume of blood is mixed in, 30 the mixture placed in the spectrometer after a fixed period of time and a reading taken. While the detailed nature of the free radical species is not yet fully understood, it has been found to have a molecular weight below 5500. This is based on the observation that using 35 HPLC (gel permeation) to obtain plasma fractions, the active species was found in the fraction having molecular weight below 5500. The esr signal is conveniently detected at a microwave frequency of 9.440 GHz with a center field of 335 Gauss. It will be appreciated that because the detection of the free radical species is at a specific frequency, the full magnetic frequency range of a conventional esr spectrometer is not necessary, thus allowing the use of a fixed magnet and considerably reducing the size and cost of a machine dedicated to the assay described herein, the aim being to quantitate signal strength as opposed to analysing signal type. The invention therefore also includes an esr spectrometer, for use in detecting and/or monitoring loss or impairment of renal function, adapted to operate over a restricted magnetic field width around the center field of the signal being detected, for example, by provision of a sweep coil to sweep 30 Gauss around the center field. The process of the present invention is applicable in several areas associated with renal malfunction. Thus it may be used to diagnose acute renal failure and, by monitoring the fall in the detected signal, the chance of patient recovery. It may also be used to monitor the effect of dialysis in patients with chronic kidney failure, thus potentially increasing the efficiency of usage of scarce dialysis unit resources. Such patients may have impaired renal function or total loss of natural renal function, relying on dialysis for survival. It will be appreciated that the ability to monitor such patients and optimize the use of dialysis can reduce dialysis time for patients and ensure that a dialysis unit is used to treat as many patients as possible. Clearance of the free radical species of uraemic plasma during haemodialysls has been investigated and found to be of the order of 80 ml/min, suggesting a molecular weight of 3000 or less. Yet again, it has been found that the detected signal can be used to monitor kidney transplant patients and predict the onset of normal function in the transplanted organ. The process can also find use in the monitoring of patients suspected of or known to have kidney impairment, for example by monitoring the patients' urine. It is envisaged that although the clinical samples will usually be taken from the patient for monitoring by esr, a continuous on-going monitor may be attached to the patient by suitable means, for example, monitoring the dialysis by analysis of either dialysate or blood. The invention will now be further described by way of example. Figure 1 shows schematically an esr spectrometer which has been modified so as to be suitable for detection of the free radical species as outlined above. The spectrometer comprises a microwave source 1, preferably solid state in order to keep the bulk of the instrument within reasonable limits. If a low voltage solid state source is employed, the source may be locked to a standard reference in the interests of minimizing cost. Microwave source 1 is linked via isolator 2 and attenuator 3 with circulator or hybrid teg 4 associated with magnet 5 with cavity 6 for introduction of the sample to be analysed. The magnet 5 need not be capable of being swept over more than a small fraction of the normal sweep range of the research spectrometers designed for a multiplicity of purposes. It is recognised that most signals obtained are for species with a g value close to the free electron value. Thus a sweep of 1-3 T is preferred, thus enabling a relatively cheap magnet to be employed. Locking to a standard reference signal is also preferred to ensure stability. Detection of the signal from the sample in cavity 6 is effected by crystal detector 7. In order to obtain good sensitivity detection this detector should be a superhet design and the final display should be geared to its analytical role, so that signal intensities are important while signal positions are secondary. Thus a comparatively inexpensive computer output may be adequate. It will be noted that the system does not require elaborate control of such functions as modulation frequency or modulation depth. Sweep times may be restricted to a selected few - very long scans (>5 sec.) are obviously not appropriate to the clinical situation. The following examples illustrate the use of an esr spectrometer in the process of the invention. Example 1. Monitoring of Patients with Chronic Renal Failure Blood was collected from patients into heparin. Blood was taken as it left the arm of the patient and not straight after passing over the dialysis membrane. For those patients on continuous ambulatory peritoneal dialysis, venous blood was taken from the antecubital fossa. All samples were centrifuged at 2000 rpm for 3 minutes. The plasma was removed and placed in a sterile tube at -70° Celsius until analysis. DBNBS was prepared from 3,5-dibromosulphanilic acid as described by Kaur i± &L, J. Chem. Soc. Che . Commun., 1981, 142-143. 10 μl of a freshly prepared solution of DBNBS (10 _1 M) was added to 190 μl phosphate buffered saline and 300 μl of plasma was added at time zero. This solution was aspirated into a round quartz capillary esr tube and placed inside the cavity of a JE0L RE IX electron spin resonance spectrometer. Center field was 335 Gauss and the microwave frequency 9.440 GHz. Esr spectra were recorded with a scan time of four minutes, and repeated until the magnitude of the spectrum remained constant. Recordings were made at room temperature. Storage of plasma at room temperature for 6 hours or at -20°C or -70 β C for up to 4 months did not cause an increment or decrement of the esr signal compared to that of fresh plasma. Plasma from normal individuals gave the signal shown in Figure 2. Plasma from patients with renal failure gave a characteristic three line spectrum (Figure 3), the activity of which was present in the fraction of serum containing molecules of molecular weight below 5500 (Figure 4). (Data using gel permeation HPLC). The addition of methanol to the system resulted in a decreased signal height. Superoxide dismutase and γ radiation had no effect. The effect of a single dialysis on the plasma activity, in a patient with stable chronic renal failure, is shown in Figure 5 over a four hour period of haemodialysls. Example 2. Monitoring of Patients with Acute Renal Failure A patient with acute renal failure, who subsequently recovered normal renal function after several periods of hae odialysis, was monitored by esr spectroscopy as described in Example 1. For comparison purposes the patient was also monitored by serum creatinine assay (measured on a SMAC autoanalyser). The results are shown in Figure 6. It will be noted that the concentration of the species detected by esr fell more rapidly and more steadily than the creatinine concentration thus giving a clearer and speedier picture that recovery of the patient was imminent and that renal function independent of creatinine clearance was improving. Example 3. Monitoring of Patients with Renal Transplants Two patients with renal transplants were monitored both by esr spectroscopy as described in Example 1 and by serum creatinine assay, measured on a SMAC autoanalyser. The results for a first patient, with a non-functioning transplant, are shown in Figure 7. The results for a second patient with a functioning transplant are shown in Figure 8.

Claims: CLAIKS 1- A process for detecting and/or monitoring loss or impairment of renal function comprising reacting a clinical sample from a patient with suspected or confirmed loss or impairment of renal function with a compound capable of interacting with a generated free radical species to form a stable radical adduct and detecting the presence of such adduct by electron spin resonance spectroscopy. 2. A process according to Claim 1 wherein the compound capable of interacting with the generated free radical species is a nitroso type compound. 3. A process according to Claim 1 wherein the compound is 3,5-dibromo-4-nitroso-benzene sulphonate. 4. A process according to any one of Claims 1 to 3 wherein the clinical sample is selected from whole blood, a fraction thereof, a dialysate or urine. 5. A process according to Claim 4 wherein the clinical sample is a plasma fraction having molecular weight below 5500. 6. 3,5-dibromo-4-nitroso-benzene sulphonate, or a related nitroso type spin trap, for use in the detection of free radical species in clinical samples from patients with suspected or confirmed loss or impairment of renal function. 7. An electron spin resonance spectrometer, suitable for use in detecting and/or monitoring loss or impairment of renal function, adapted to operate over a restricted magnetic field width around the center field of the signal being detected. 8. A spectrometer according to Claim 7 which is adapted to operate over a restricted magnetic field width around a center field of 335 Gauss.

Inventors: BLAKE DAVID RUSSELL, NAZHAT NAJDAT BADI, ROSELAAR SIMON EDWIN, WINYARD PAUL GRAHAM, BLAKE, DAVID, RUSSELL, NAZHAT, NAJDAT, BADI, ROSELAAR, SIMON, EDWIN, WINYARD, PAUL, GRAHAM
Applicants: BRITISH TECH GROUP, BRITISH TECHNOLOGY GROUP LTD.
Application year: 1992
Main classification: G01R33
IPC codes: A61B5, A61B10, A61K49, G01N33, G01R33