1 H -NMR Spectroscopy as a Means of Monitoring Nephrotoxicity as Exemplified by Studies with Cephaloridine L. B. Murgatroyd, R. J. Middleton ICI Pharmaceuticals 4TG, UK
Safety
Pickford,
of Medicines
I. K.
B. J.
Smith, I. D. Wilson &
Department, Mereside. Alderley Park, Macclesfield,
SK10
1 Male albino rats were dosed intravenously with either 0.9% saline or cephaloridine in -1 for 7 d. saline at doses of 650, 750 or 950 mg kg -1 d 2 Urine analysis on day 3, after two doses of cephaloridine showed dose-related increases in glucose, total protein, N-acetyl β-D-glucosaminidase, y-glutamyltranspeptidase, alkaline H-NMR spectroscopy showed corresponding phosphatase and lactate dehydrogenase. 1 disturbed profiles of products of intermediary metabolism indicative of a disruption of renal function. H-NMR and conventional 3 By day 6, after five doses of cephaloridine, analysis by both 1 H-NMR was demonstrated to methods showed that all indices had returned to normal.1 provide useful complementary information to conventional techniques on the time course of the onset of the nephrotoxicity and the recovery phase, and was at least as sensitive as conventional urine analysis.
Introduction
(’H) nuclear magnetic reson(NMR) spectroscopy is emerging as a powerful technique for the analysis of biological fluids (e.g., plasma, urine and bile etc.). Applications are wide ranging and include clinical chemistry, drug metabolism and toxicology. 1,2 This is the result of the ability of ’H-NMR to provide rapid analysis of solutes of widely different physical and chemical properties present in the same sample with equal ease. Indeed, in principle, providing that a molecule contains protons, the only criterion which must be set to enable it’s detection by ’H-NMR in a sample
High
field proton
ance
such as urine, is that it must be present at a concentration of 50 pm or greater. In practice this means that in the ’H-NMR analysis of a biological fluid such as urine, a characteristic fingerprint is provided which enables the simultaneous qualitative analysis of Krebs cycle intermediates such as citrate, succinate, and aketoglutarate as well as amino acids, organic acids, glucose, acetate, creatinine, hippuric acid and any drug-related material present. This type of analysis can reveal, very simply and rapidly, any perturbation of the normal pattern of signals following a toxic insult. In addition, the presence of new resonances in the biofluid spectrum may
information on the site and mode of action of the toxin.3 Indeed, recent work using a range of nephro- and hepato-toxins has clearly shown that compounds with the same tissue site of action produce similar disturbances in the normal urinary fingerprint. 3,4 This work has since been extended by the use of sophisticated computer-based pattern recognition methods in4 order to classify the types of toxicity produced.4 In general, however, the bulk of the studies performed to date using ’H-NMR as a means of investigating organ-specific toxicity have been short-term, single dose, experiments. In order to demonstrate the potential of this methodology as a non-invasive technique for monitoring longer term studies ’H-NMR has been used to monitor the effects of daily administration of cephaloridine, a known nephrotoxin,5 at various dose intervals. In addition the results of H-NMR are compared with those obtained by conventional techniques normally used in such studies.
provide
’
Materials and methods
Cephaloridine
was
supplied by
Glaxo Pharma-
ceuticals, 891/995 Greenford Road, Greenford,
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
.
36 L
Middlesex and was 96% pure as assessed by ’HNMR. The dosing solution was prepared by dissolving the drug in 0.9% aqueous sodium chloride at a nominal concentration of 200 mg
mi-I. The study was performed using 12 healthy male ICI bred APfSD Wistar derived rats which were housed individually in metabolism cages. The animals were acclimatised for 2 d prior to dosing, and water and food were available ad libitum throughout the study. The rats (three per dose group) received either saline or cephaloridine intravenously at doses of 650, 750 or 950 mg kg-’ d-’ for 7 d. The bodyweight of the animals was recorded before dosing and then daily for the remainder of the study. Food and water consumption were recorded. Complete 24-h urine samples were collected from all anin,als for the pre-study period and daily throughout the study. Urinary volumes were determined by weighing (assuming a relative density of 1.0).. Samples taken on days 3 and 6 of the study (after two and five doses of cephaloridine), were analysed quantitatively for glucose, creatinine, sodium, potassium and total protein. The volume, specific gravity and osmolality of these samples were also determined. In addition, the activities of the following enzymes were estimated; alkaline phosphatase
priate because of the large variability associated with increases in some means. Instead, the ranks were analysed using a one-way analysis of variance followed by a Student’s t-test to compare each group with the control. A probability of less than 0.01 was considered to indicate a significant difference from the control value. Results No
significant differences were observed in weight gain or food consumption between control and dosed animals. Increased water consumption and urine production were recorded for animals dosed at 950 mg kg-’ d-’ between days 2 and 5 of the study when compared to controls (see Figures la and b). A typical ’H-NMR spectrum from a control animal is shown in Figure 2. Prominent resonances for succinate, citrate, creatinine, allantoin, hippurate and other compounds normally excreted in urine were present. A series of spectra .from
(ALP), N-acetyl J3-~-glucosaminidase (NAG), y-glutamyltranspeptidase (GGT) and lactatc dehydrogenase (LDH). All urine samples from animals dosed at 950 mg kg-’ d-1 were analysed qualitatively using’HNMR, whilst those samples taken from animals dosed at 650 and 750 mg kg-1 d-1 were analysed on days 3 and 7 only. Aliquots of urine (1.6 ml) were taken and mixed with 5 M urea in 2H20 (400 Ill) and the pH adjusted to 3.5. ’H-NMR was performed using the WATR water suppression technique as modified by Connor et al.6 All spectra were recorded at ambient probe temperature on a Bruker AM 400 NMR operating at 9.4 tesla (400
spectrometer
MHz). Spectra were the result of 64 free induction decays which were collected into 32 x 1024 data points. All animals were necropsied on day 8. The kidneys were removed from all the animals at necropsy and their weights were determined following removal of excess fat and prior to fixation. The kidneys were then preserved in 10% buffered formalin, processed through paraffin wax and sectioned. Sections from each kidney were stained with haematoxylin and eosin prior to histopathological examination. The mean and standard deviation were calculated for each parameter in each group. Statistical analysis of the raw data was considered inappro-
Figure la Group mean daily water intake of control (A) and animals dosed intravenously with 650 (8), 750 (S) or 950 (0) mg kg-1 d-’ of cephaloridine. b Group mean daily urine production of control (A) and animals dosed intravenously with 650 (0), 750 (S) or 950 (0) mg kg-’ d-’ of cephaloridine. Days -2 and -1 represent days prior to dosing. Day 1 immediately follows day -1; there is no day 0.
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
37
2 A typical ’H-NMR spectrum of control rat urine. Key: At, Allantoin; a-Kg; a - Ketoglutarate; cit; citrate; Cn; creatinineficr; creatine; DMA; dimethylamine; Hip; hippurate; Suc; succinate; TMAO; trimethyl-
Figure
amine N-oxide.
the urine of one animal dosed with cephaloridine mg kg-’ d-’, after the first, second and third doses (days 2, 3 and 4, respectively) are shown in Figures 3a-3c. The day 2 spectrum (Figure 3a) shows, in addition to the normal endogenous components, very prominent resonances for cephaloridine. However, the spectrum obtained for day 3 shows a grossly disturbed pattern (Figure 3b). Thus, in addition to the cephaloridine-related signals, large quantities of glucose were present, together with resonances for the amino acids, valine, alanine and glutamine. High concentrations of lactate were also noted whilst hippurate was absent. By day 4, however, (Figure 3c) concentrations of glucose were falling and the normal pattern of endogenous metabolites were being restored. Normalization of the urinary profile continued over days 5 and 6, and by day 7 the NMR spectra of urine samples from these animals were normal except for signals resulting from the presence of cephaloridine. In the case of the lowest dose used in the study (650 mg kg~’ d-’) a generally normal pattern of endogenous components was observed for samples taken on day 3 of the study. Small amounts of valine, lactate, alanine and glucose etc. were detected, but the perturbation of the normal profile was small compared to the results obtained at 950 mg kg-1 d-’. at 950
At 750 mg kg-1 d-’ the NMR spectra obtained day 3 of the study showed a pattern not dissimilar to that obtained at 950 mg d-1, although somewhat less severe. Typical spectra for animals dosed at 650, 750 and 950 mg kg-1 d-1 are shown in Figure 4 for comparison. By day 7 the NMR spectra of all rats dosed with cephaloridine were normal with regard to endogenous
on
kg’
metabolites. The
results
of
conventional
methods
of
urinalysis for various factors including volume, osmolality, total protein, potassium, sodium and glucose concentrations and urinary enzymes are shown in Table 1. These results show that urine production increased with dose and remained
high (Figure lb). Osmolality fell
with increasing dose, but returned to normal by day 6 of the’ study. The rise and fall om urinary glucose concentrations observed in the ’H-NMR spectra of urine samples from the highest dose groups over days 2 to 4 were confirmed by specific analysis and reached 114 ± 19.8 mmol 1-1 on day 3 for animals dosed at 950 mg kg-1 d-’ . By day 6, the glucose concentrations for all the animals dosed with cephaloridine were of the same order as the controls. In addition, the measurement of total urinary protein and the enzymes NAG, GGT, ALP and LDH all showed an increase with increasing dose of cephaloridine
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
38
3 Tvpical ’H-NMR spectra of urine obtained for days 2 (a), 3 (b) and 4 (c) of the study from animals dosed at 950 mg kg-’ d-’ (after the first. second and third doses. respectively). Key as for Figure 2 with in addition: Ala: alanine: Glu: ~~lutamine; Gluc. glucose: Lac: lactate: Lvs: Ivsine: Val: valine: u and fl alpha and beta anomeric protons of glucose. respectively. Ceph - resonances arising from cephaloridine.
Figure
=
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
39
Figure 4 Typical 1H-NMR spectra of urine obtained from day 3 of the study (after two doses) from animals dosed at 650 (a), 750 (b) and 950 (c) mg kg 1 d-I. Key as for Figures 2 and 3.
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
40
Table 1 Group mean with cephaloridine at
**
Significance level Day 3 sampled *
=
(± s.d.) urinalysis data obtained from days 3 and 6 of the study for rats dosed intravenously 0, 650, 750
on
level
*
950 mg
P < 0.01, n 3. after two doses. Day 6
* P
kg-’
d-’ for 7 d.
=
Table 2 Mean kidney weight and with cephaloridine at 0, 650, 750
Significance
or
Safety
Pickford,
of Medicines
I. K.
B. J.
Smith, I. D. Wilson &
Department, Mereside. Alderley Park, Macclesfield,
SK10
1 Male albino rats were dosed intravenously with either 0.9% saline or cephaloridine in -1 for 7 d. saline at doses of 650, 750 or 950 mg kg -1 d 2 Urine analysis on day 3, after two doses of cephaloridine showed dose-related increases in glucose, total protein, N-acetyl β-D-glucosaminidase, y-glutamyltranspeptidase, alkaline H-NMR spectroscopy showed corresponding phosphatase and lactate dehydrogenase. 1 disturbed profiles of products of intermediary metabolism indicative of a disruption of renal function. H-NMR and conventional 3 By day 6, after five doses of cephaloridine, analysis by both 1 H-NMR was demonstrated to methods showed that all indices had returned to normal.1 provide useful complementary information to conventional techniques on the time course of the onset of the nephrotoxicity and the recovery phase, and was at least as sensitive as conventional urine analysis.
Introduction
(’H) nuclear magnetic reson(NMR) spectroscopy is emerging as a powerful technique for the analysis of biological fluids (e.g., plasma, urine and bile etc.). Applications are wide ranging and include clinical chemistry, drug metabolism and toxicology. 1,2 This is the result of the ability of ’H-NMR to provide rapid analysis of solutes of widely different physical and chemical properties present in the same sample with equal ease. Indeed, in principle, providing that a molecule contains protons, the only criterion which must be set to enable it’s detection by ’H-NMR in a sample
High
field proton
ance
such as urine, is that it must be present at a concentration of 50 pm or greater. In practice this means that in the ’H-NMR analysis of a biological fluid such as urine, a characteristic fingerprint is provided which enables the simultaneous qualitative analysis of Krebs cycle intermediates such as citrate, succinate, and aketoglutarate as well as amino acids, organic acids, glucose, acetate, creatinine, hippuric acid and any drug-related material present. This type of analysis can reveal, very simply and rapidly, any perturbation of the normal pattern of signals following a toxic insult. In addition, the presence of new resonances in the biofluid spectrum may
information on the site and mode of action of the toxin.3 Indeed, recent work using a range of nephro- and hepato-toxins has clearly shown that compounds with the same tissue site of action produce similar disturbances in the normal urinary fingerprint. 3,4 This work has since been extended by the use of sophisticated computer-based pattern recognition methods in4 order to classify the types of toxicity produced.4 In general, however, the bulk of the studies performed to date using ’H-NMR as a means of investigating organ-specific toxicity have been short-term, single dose, experiments. In order to demonstrate the potential of this methodology as a non-invasive technique for monitoring longer term studies ’H-NMR has been used to monitor the effects of daily administration of cephaloridine, a known nephrotoxin,5 at various dose intervals. In addition the results of H-NMR are compared with those obtained by conventional techniques normally used in such studies.
provide
’
Materials and methods
Cephaloridine
was
supplied by
Glaxo Pharma-
ceuticals, 891/995 Greenford Road, Greenford,
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
.
36 L
Middlesex and was 96% pure as assessed by ’HNMR. The dosing solution was prepared by dissolving the drug in 0.9% aqueous sodium chloride at a nominal concentration of 200 mg
mi-I. The study was performed using 12 healthy male ICI bred APfSD Wistar derived rats which were housed individually in metabolism cages. The animals were acclimatised for 2 d prior to dosing, and water and food were available ad libitum throughout the study. The rats (three per dose group) received either saline or cephaloridine intravenously at doses of 650, 750 or 950 mg kg-’ d-’ for 7 d. The bodyweight of the animals was recorded before dosing and then daily for the remainder of the study. Food and water consumption were recorded. Complete 24-h urine samples were collected from all anin,als for the pre-study period and daily throughout the study. Urinary volumes were determined by weighing (assuming a relative density of 1.0).. Samples taken on days 3 and 6 of the study (after two and five doses of cephaloridine), were analysed quantitatively for glucose, creatinine, sodium, potassium and total protein. The volume, specific gravity and osmolality of these samples were also determined. In addition, the activities of the following enzymes were estimated; alkaline phosphatase
priate because of the large variability associated with increases in some means. Instead, the ranks were analysed using a one-way analysis of variance followed by a Student’s t-test to compare each group with the control. A probability of less than 0.01 was considered to indicate a significant difference from the control value. Results No
significant differences were observed in weight gain or food consumption between control and dosed animals. Increased water consumption and urine production were recorded for animals dosed at 950 mg kg-’ d-’ between days 2 and 5 of the study when compared to controls (see Figures la and b). A typical ’H-NMR spectrum from a control animal is shown in Figure 2. Prominent resonances for succinate, citrate, creatinine, allantoin, hippurate and other compounds normally excreted in urine were present. A series of spectra .from
(ALP), N-acetyl J3-~-glucosaminidase (NAG), y-glutamyltranspeptidase (GGT) and lactatc dehydrogenase (LDH). All urine samples from animals dosed at 950 mg kg-’ d-1 were analysed qualitatively using’HNMR, whilst those samples taken from animals dosed at 650 and 750 mg kg-1 d-1 were analysed on days 3 and 7 only. Aliquots of urine (1.6 ml) were taken and mixed with 5 M urea in 2H20 (400 Ill) and the pH adjusted to 3.5. ’H-NMR was performed using the WATR water suppression technique as modified by Connor et al.6 All spectra were recorded at ambient probe temperature on a Bruker AM 400 NMR operating at 9.4 tesla (400
spectrometer
MHz). Spectra were the result of 64 free induction decays which were collected into 32 x 1024 data points. All animals were necropsied on day 8. The kidneys were removed from all the animals at necropsy and their weights were determined following removal of excess fat and prior to fixation. The kidneys were then preserved in 10% buffered formalin, processed through paraffin wax and sectioned. Sections from each kidney were stained with haematoxylin and eosin prior to histopathological examination. The mean and standard deviation were calculated for each parameter in each group. Statistical analysis of the raw data was considered inappro-
Figure la Group mean daily water intake of control (A) and animals dosed intravenously with 650 (8), 750 (S) or 950 (0) mg kg-1 d-’ of cephaloridine. b Group mean daily urine production of control (A) and animals dosed intravenously with 650 (0), 750 (S) or 950 (0) mg kg-’ d-’ of cephaloridine. Days -2 and -1 represent days prior to dosing. Day 1 immediately follows day -1; there is no day 0.
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
37
2 A typical ’H-NMR spectrum of control rat urine. Key: At, Allantoin; a-Kg; a - Ketoglutarate; cit; citrate; Cn; creatinineficr; creatine; DMA; dimethylamine; Hip; hippurate; Suc; succinate; TMAO; trimethyl-
Figure
amine N-oxide.
the urine of one animal dosed with cephaloridine mg kg-’ d-’, after the first, second and third doses (days 2, 3 and 4, respectively) are shown in Figures 3a-3c. The day 2 spectrum (Figure 3a) shows, in addition to the normal endogenous components, very prominent resonances for cephaloridine. However, the spectrum obtained for day 3 shows a grossly disturbed pattern (Figure 3b). Thus, in addition to the cephaloridine-related signals, large quantities of glucose were present, together with resonances for the amino acids, valine, alanine and glutamine. High concentrations of lactate were also noted whilst hippurate was absent. By day 4, however, (Figure 3c) concentrations of glucose were falling and the normal pattern of endogenous metabolites were being restored. Normalization of the urinary profile continued over days 5 and 6, and by day 7 the NMR spectra of urine samples from these animals were normal except for signals resulting from the presence of cephaloridine. In the case of the lowest dose used in the study (650 mg kg~’ d-’) a generally normal pattern of endogenous components was observed for samples taken on day 3 of the study. Small amounts of valine, lactate, alanine and glucose etc. were detected, but the perturbation of the normal profile was small compared to the results obtained at 950 mg kg-1 d-’. at 950
At 750 mg kg-1 d-’ the NMR spectra obtained day 3 of the study showed a pattern not dissimilar to that obtained at 950 mg d-1, although somewhat less severe. Typical spectra for animals dosed at 650, 750 and 950 mg kg-1 d-1 are shown in Figure 4 for comparison. By day 7 the NMR spectra of all rats dosed with cephaloridine were normal with regard to endogenous
on
kg’
metabolites. The
results
of
conventional
methods
of
urinalysis for various factors including volume, osmolality, total protein, potassium, sodium and glucose concentrations and urinary enzymes are shown in Table 1. These results show that urine production increased with dose and remained
high (Figure lb). Osmolality fell
with increasing dose, but returned to normal by day 6 of the’ study. The rise and fall om urinary glucose concentrations observed in the ’H-NMR spectra of urine samples from the highest dose groups over days 2 to 4 were confirmed by specific analysis and reached 114 ± 19.8 mmol 1-1 on day 3 for animals dosed at 950 mg kg-1 d-’ . By day 6, the glucose concentrations for all the animals dosed with cephaloridine were of the same order as the controls. In addition, the measurement of total urinary protein and the enzymes NAG, GGT, ALP and LDH all showed an increase with increasing dose of cephaloridine
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
38
3 Tvpical ’H-NMR spectra of urine obtained for days 2 (a), 3 (b) and 4 (c) of the study from animals dosed at 950 mg kg-’ d-’ (after the first. second and third doses. respectively). Key as for Figure 2 with in addition: Ala: alanine: Glu: ~~lutamine; Gluc. glucose: Lac: lactate: Lvs: Ivsine: Val: valine: u and fl alpha and beta anomeric protons of glucose. respectively. Ceph - resonances arising from cephaloridine.
Figure
=
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
39
Figure 4 Typical 1H-NMR spectra of urine obtained from day 3 of the study (after two doses) from animals dosed at 650 (a), 750 (b) and 950 (c) mg kg 1 d-I. Key as for Figures 2 and 3.
Downloaded from het.sagepub.com at UNIV CALIFORNIA SANTA BARBARA on June 25, 2015
40
Table 1 Group mean with cephaloridine at
**
Significance level Day 3 sampled *
=
(± s.d.) urinalysis data obtained from days 3 and 6 of the study for rats dosed intravenously 0, 650, 750
on
level
*
950 mg
P < 0.01, n 3. after two doses. Day 6
* P
kg-’
d-’ for 7 d.
=
Table 2 Mean kidney weight and with cephaloridine at 0, 650, 750
Significance
or
