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Proton nuclear magnetic resonance spectroscopy of urine for rapid multicomponent analysis of intraoperative cellular metabolites
Clinica Chimica Acta 279 (1999) 117–124
Proton nuclear magnetic resonance spectroscopy of urine for rapid multicomponent analysis of intraoperative cellular metabolites a, a b c Tsuneo Tatara *, Yasuhide Iwao , Junzo Takeda , Yoshimasa Ishihara , Shoichi Ohkochi c , Hisashi Uedaira c a
Department of Anesthesiology, School of Medicine, Kyorin University, 6 -20 -2 Shinkawa, Mitaka-shi, 181 -8611 Tokyo, Japan b Department of Anesthesiology, School of Medicine, Keio University, Tokyo, Japan c Department of Materials Chemistry, College of Engineering, Hosei University, Tokyo, Japan Accepted 8 October 1998
Abstract We serially measured the proton nuclear magnetic resonance spectra of the urine of intraoperative patients over time to assess their potential use for rapid multicomponent analysis of cellular metabolites. Within a few minutes, the spectra provided signals of many low molecular weight urinary metabolites, including amino acids, ketone bodies, lactate, and glucose. The proton magnetic resonance spectra of the urine of most of the intraoperative patients showed large increases in urinary excretion of alanine, ketone bodies, and lactate and / or glucose, confirming alterations in energy substrate–endocrine relationships during the perioperative period. These metabolic changes appeared to be roughly proportional to the degree of surgical stress, but they were not consistent among patients who underwent the same operation. The study suggests that routine intraoperative metabolic monitoring is necessary to prevent critical metabolic disorders and that proton nuclear magnetic resonance spectroscopy of urine may meet this need by allowing rapid multicomponent analysis of cellular metabolites. 1999 Elsevier Science B.V. All rights reserved. Keywords: Nuclear magnetic resonance; Metabolism; Proton; Surgery; Urine
*Corresponding author. 0009-8981 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00174-0
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T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
1. Introduction Surgical stress induces major changes in energy substrate–endocrine relationships that may result in critical metabolic derangements [1,2]. However, routine analysis of body fluids that reflect these cellular metabolic changes has been limited during surgery, because conventional clinical assays such as enzymatic techniques are time-consuming and can analyze few substances in a single assay. Proton nuclear magnetic resonance ( 1 H NMR) spectroscopy allows rapid multicomponent analysis of low molecular weight compounds in solution [3,4]. 1 H nuclei in samples placed in a magnetic environment have a specific resonant frequency and give rise to a unique line on the chemical shift axis. The signal intensity is proportional to the number of 1 Hs and enables compounds to be analyzed quantitatively. During the past decade, 1 H NMR measurements of body fluids such as serum, urine, synovial fluid, and cerebrospinal fluid have been successfully used as an aid to metabolic monitoring and disease screening [3–6]. Since many of the urinary metabolites of interest perioperatively are small, 1 H-containing molecules, 1 H NMR measurement of urine is expected to provide detailed insight into intraoperative cellular metabolism. In this study we assessed its potential by serially measuring the 1 H NMR spectra of urine during surgery.
1.1. Patients and methods This study was conducted on seven patients scheduled for surgery with an expected operative time greater than 3 h. Patients with preexisting metabolic disorders were excluded. Written consent was obtained from each patient before entering the study, and the study protocol was approved by the institutional ethics committee. The patients urinated an hour before the incision was made. Anesthesia was maintained with nitrous oxide and isoflurane or sevoflurane, and acetated Ringer’s solution was infused during anesthesia. Urine samples were collected from each patient through an indwelling urinary bladder catheter and their volume was measured every hour from the time anesthesia was induced until recovery from anesthesia.
1.2. 1 H NMR urinalysis The urine was collected in polypropylene tubes, promptly frozen in liquid nitrogen, and stored at 2 308C until used. A 50-ml volume of 2 H 2 0 was added to 0.5 ml of urine samples for field / frequency locking. Sodium tetradeutero-3trimethyl-silylpropionate (TSP), 5.8 mmol / l, was dissolved in the 2 H 2 0 to serve
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
119
as a reference for the chemical shifts and peak areas. NMR measurements were made on an FT-NMR spectrometer (Jeol EX270, Jeol, Tokyo, Japan) operating at 270.05 MHz in the homogated decoupling mode. All spectra were recorded at 258C. The spectral conditions were: spectral width 5405.4 Hz, acquisition time 3.28 s, pulse width 5.0 ms, delay 5.00 s, and transient 40. The signal for H 2 0 near 4.8 ppm was reduced by the presaturation method. The signal assignments were referred to the chemical shifts of metabolites previously reported [3–6]. Urinary concentrations of alanine, ketone bodies (3-hydroxybutyrate, acetone and acetoacetate) and lactate were estimated from the ratios of their peak areas to those of TSP. Relative concentrations of glucose were obtained as the relative peak heights at 3.7 ppm to those of TSP because of their multiple peaks. The urinary excretion rates of these metabolites were calculated thus: urinary excretion rate of the metabolites (mmol ? kg 21 ? h 21 ) 5 urinary concentration of the metabolites (mmol ? l 21 ) urine output per hour (l ? h 21 ) / body weight (kg)
2. Results Patient characteristics are shown in Table 1. Three of the seven patients underwent gastrectomy, two underwent mastectomy, and two underwent minor surgery. All patients recovered from anesthesia without apparent metabolic complications such as consciousness disturbances.
2.1. Signal assignments Fig. 1 shows a typical 1 H NMR spectrum of urine from a healthy volunteer. The spectrum was complex, with many signals being detected within the few minutes of total scanning time. A wide range of urinary metabolites could be identified in the chemical shift range of 1–4.2 ppm which is consistent with previous reports [3–5], but many minor resonances from metabolites in low Table 1 Characteristics of the patients Case a
Age (years) / gender
Operation
Operative time (h)
G1 G2 G3 M1 M2 T L
52 / M 49 / M 60 / F 60 / F 56 / F 29 / M 22 / M
Total gastrectomy Total gastrectomy Total gastrectomy Radical mastectomy Radical mastectomy Construction of tympanum Repair of cleft lip
5 4.5 3.5 4 4 5 4
a
Initials represent the operation.
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T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
Fig. 1. A typical 1 H NMR spectrum of urine from a healthy volunteer (26 year old male). Ala, alanine; Bu, 3-hydroxybutyrate; Ch, choline; Cit, citrate; Cn, creatinine; DMA, dimethylamine; Glu, glucose; Gly, glycine; Lac, lactate; N-Ac, N-acetylated glycoprotein; TMAO, trimethylamineN-oxide.
concentrations remain to be assigned. The strongest signals, at 3.05 and 4.08 ppm, were from creatinine, and the peaks at 3.23, 3.29 and 3.57 ppm were assignable to choline, trimethylamine-N-oxide, and glycine, respectively. Glucose showed multiple peaks in the relatively crowded region of the spectrum between 3.2 and 4.0 ppm. The small double peaks at 1.25, 1.33, and 1.47 ppm have been assigned to 3-hydroxybutyrate, lactate, and alanine, respectively. N-Acetylated glycoprotein gave rise to a single peak at 2.04 ppm. Double peaks around 2.6 ppm were assigned to citrate, and dimethylamine showed a single peak at 2.73 ppm.
2.2. Serial 1 H NMR spectra and urinary excretion rates of metabolites Fig. 2 shows the time course of the 1 H NMR spectra of urine collected in case G2. The 1 H NMR spectra did not change significantly during the first hour after the incision, but the 3-hydroxybutyrate and acetone signals appeared after the end of the first hour. The most remarkable changes were that the signal intensities of lactate and glucose significantly increased 2 h after the incision and throughout the rest of the surgery. The creatinine signals did not show significant changes at any time during surgery. The time-course changes in the urinary excretion rates of metabolites in each
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
121
Fig. 2. Time-course of the 1 H NMR spectra of urine from case G2. Time is shown in hours post-incision. Urine outputs per hour were 200, 50, 70, 60, 50, 60 ml (from the top). An, acetone; Bu, 3-hydroxybutyrate; Cn, creatinine; Glu, glucose; Lac, lactate.
patient are summarized in Fig. 3. The rates of alanine excretion significantly increased over 2–3 h in cases G2, G3, and M2. The 3-hydroxybutyrate and acetone excretion rates appeared to increase during the latter half of surgery in
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T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
Fig. 3. Time-course changes in the urinary excretion rates of metabolites in each patient. * Relative value.
cases G1, G2, G3, M1, and T. Acetoacetate showed significant increases over 1–2 h in cases G2, G3, and M1, and at 4–5 h in case G1. Lactate significantly increased at 2–3 h in cases G2 and M2, and further increased throughout the remainder of surgery in case G2. Cases G2 and M2 showed marked increases in glucose at 3–4 and 2–3 h, respectively. Case L did not show significant changes in the excretion rates of metabolites at any time during surgery except small increases of glucose at 0–1 and 1–2 h.
3. Discussion The NMR spectra of urine from most of the intraoperative patients showed remarkable increases in urinary excretion of alanine, ketone bodies, and lactate, and / or glucose. These metabolic profiles confirm the energy substrate–endocrine alterations that occur during surgery [1]. The increased excretion of alanine may be explained by accelerated protein catabolism for gluconeogenesis induced by surgical trauma. The appearance of ketone bodies is probably associated with the release of fatty acids as a fuel source. Glycogenolysis in muscle and / or decreased tissue perfusion may explain the increased lactate excretion. The increased glucose excretion may be caused by increased hepatic glucose output and / or the reduced peripheral use of glucose known as ‘diabetes of trauma’. These metabolic changes appeared to be roughly proportional to the degree of surgical stress, since patients T and L, who underwent minor surgery, showed
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
123
small changes in the urinary excretion of the metabolites. However, they were not consistent among the patients who underwent same operation: case G2 showed increased excretion of all urinary metabolites, while the rates of excretion of alanine and ketone bodies increased in case G3, and ketone bodies alone increased in case G1. It is also noteworthy that these metabolic changes became prominent 2 or 3 h after the incision was made, a finding consistent with the results of our previous study on serum metabolites [7]. The increased excretion of urinary metabolites during surgery tended to return to preoperative levels at the end of surgery, but in some cases it remained increased or rather enhanced at the end of surgery. The time course of 1 H NMR spectra thus allows close monitoring of the pathological status of patients during all phases of surgery and recovery. In addition, these findings demonstrate that intraoperative metabolic alterations are difficult to anticipate, and therefore routine intraoperative monitoring of cellular metabolites especially their time course is necessary to obviate critical metabolic disorders. The current study demonstrates two unique advantages of 1 H NMR spectroscopy of urine for perioperative metabolic monitoring. First, it allows rapid multicomponent analysis of urinary metabolites. The detection of a wide range of metabolites is of particular clinical usefulness when unchecked metabolic alterations due to preoperative starvation and surgical trauma have the potential to develop into critical metabolic derangements. In addition, almost all real-time metabolic monitoring is attractive, because surgeons are sometimes obliged to change the scheduled operative procedure to a simpler and less stressful one in the face of life-threatening metabolic disorders such as severe ketosis, hyperglycemia, and / or lactic acidosis. Another advantage is that the NMR spectra patterns of energy substrates, such as amino acids, ketone bodies, lactate, and glucose provide information on perioperative fuel metabolism characterized as the catabolic state [1]. For example, the NMR spectrum patterns of urinary amino acids as well as glucose may be helpful in deciding the optimal intraoperative dose of glucose as a fuel source, which is supposed to suppress protein catabolism [1]. In the routine application of 1 H NMR spectroscopy of urine for cellular metabolic monitoring, some problems must be resolved. 1 H NMR spectroscopy is a relatively insensitive method capable of detecting species at around 0.1 mmol / l, with precise quantification being uncommon because of signal line broadening or overlapping [3,4]. Nevertheless, once metabolites showing abnormal excretion profiles are detected in the wide screen of the 1 H NMR spectrum, appropriate evaluation and therapy for this abnormality can be instituted, and more sensitive analysis then can be readily performed saving cost and time for unnecessary specific tests. It is true that the high-field NMR instruments are expensive, but their convenience and cost effectiveness is also
124
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
valuable because their maintenance costs are low and instruments can process samples around the clock in an automated manner [4]. The NMR spectra should be carefully analyzed considering confounding variables such as drugs which may influence NMR spectra. This problem may be resolved by obtaining the NMR signals of drugs alone and measuring time course of the NMR spectra from the administration of the drugs. The variations of the NMR spectra within normals are unlikely to become a serious confounding factor as long as the time course of the NMR spectra is discussed. Although the utility of 1 H NMR spectroscopy of urine for metabolic monitoring has not been widely appreciated by clinicians, our results suggest that 1 H NMR spectroscopy of urine, especially the time-course findings, provide a novel approach to cellular metabolic monitoring during surgery.
References [1] Biebuyck JF, Stene JK. Nutritional aspects. In: Miller RD, editor. Anesthesia, 3rd ed. New York: Churchill Livingstone, 1990:2277–2305. [2] Tonnesen AS. Monitoring the kidney and urine. In: Blitt CD, editor. Monitoring in Anesthesia and Critical Care Medicine, 2nd ed. New York: Churchill Livingstone, 1990:575– 594. [3] Nicholson JK, Wilson I. High resolution proton magnetic resonance spectroscopy of biological fluids. Prog NMR Spectrosc 1989;21:449–501. [4] Reglinski J, Watson ID. An analytical perspective of NMR spectroscopy of biological fluids and cells. Ann Clin Biochem 1996;33:290–307. [5] Foxall PJD, Mellotte GJ, Bending MR, Lindon JC, Nicholson JK. NMR spectroscopy as a novel approach to the monitoring of renal transplant function. Kidney Int 1993;43:234–45. [6] Sweatman BC, Farrant RD, Holmes E, Ghauri FY, Nicholson JK, Lindon JC. 600 MHz 1 H-NMR spectroscopy of human cerebrospinal fluid: effects of sample manipulation and assignment of resonances. J Pharm Biomed Anal 1993;11:651–64. [7] Tatara T, Noguchi J, Fukushima K, Ishihara Y, Ohkouchi S, Uedaira H. Proton NMR spectroscopic studies of serum as an aid to perioperative cellular metabolic monitoring. Physiol Chem Phys Med NMR 1995;27:121–9.
Proton nuclear magnetic resonance spectroscopy of urine for rapid multicomponent analysis of intraoperative cellular metabolites a, a b c Tsuneo Tatara *, Yasuhide Iwao , Junzo Takeda , Yoshimasa Ishihara , Shoichi Ohkochi c , Hisashi Uedaira c a
Department of Anesthesiology, School of Medicine, Kyorin University, 6 -20 -2 Shinkawa, Mitaka-shi, 181 -8611 Tokyo, Japan b Department of Anesthesiology, School of Medicine, Keio University, Tokyo, Japan c Department of Materials Chemistry, College of Engineering, Hosei University, Tokyo, Japan Accepted 8 October 1998
Abstract We serially measured the proton nuclear magnetic resonance spectra of the urine of intraoperative patients over time to assess their potential use for rapid multicomponent analysis of cellular metabolites. Within a few minutes, the spectra provided signals of many low molecular weight urinary metabolites, including amino acids, ketone bodies, lactate, and glucose. The proton magnetic resonance spectra of the urine of most of the intraoperative patients showed large increases in urinary excretion of alanine, ketone bodies, and lactate and / or glucose, confirming alterations in energy substrate–endocrine relationships during the perioperative period. These metabolic changes appeared to be roughly proportional to the degree of surgical stress, but they were not consistent among patients who underwent the same operation. The study suggests that routine intraoperative metabolic monitoring is necessary to prevent critical metabolic disorders and that proton nuclear magnetic resonance spectroscopy of urine may meet this need by allowing rapid multicomponent analysis of cellular metabolites. 1999 Elsevier Science B.V. All rights reserved. Keywords: Nuclear magnetic resonance; Metabolism; Proton; Surgery; Urine
*Corresponding author. 0009-8981 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00174-0
118
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
1. Introduction Surgical stress induces major changes in energy substrate–endocrine relationships that may result in critical metabolic derangements [1,2]. However, routine analysis of body fluids that reflect these cellular metabolic changes has been limited during surgery, because conventional clinical assays such as enzymatic techniques are time-consuming and can analyze few substances in a single assay. Proton nuclear magnetic resonance ( 1 H NMR) spectroscopy allows rapid multicomponent analysis of low molecular weight compounds in solution [3,4]. 1 H nuclei in samples placed in a magnetic environment have a specific resonant frequency and give rise to a unique line on the chemical shift axis. The signal intensity is proportional to the number of 1 Hs and enables compounds to be analyzed quantitatively. During the past decade, 1 H NMR measurements of body fluids such as serum, urine, synovial fluid, and cerebrospinal fluid have been successfully used as an aid to metabolic monitoring and disease screening [3–6]. Since many of the urinary metabolites of interest perioperatively are small, 1 H-containing molecules, 1 H NMR measurement of urine is expected to provide detailed insight into intraoperative cellular metabolism. In this study we assessed its potential by serially measuring the 1 H NMR spectra of urine during surgery.
1.1. Patients and methods This study was conducted on seven patients scheduled for surgery with an expected operative time greater than 3 h. Patients with preexisting metabolic disorders were excluded. Written consent was obtained from each patient before entering the study, and the study protocol was approved by the institutional ethics committee. The patients urinated an hour before the incision was made. Anesthesia was maintained with nitrous oxide and isoflurane or sevoflurane, and acetated Ringer’s solution was infused during anesthesia. Urine samples were collected from each patient through an indwelling urinary bladder catheter and their volume was measured every hour from the time anesthesia was induced until recovery from anesthesia.
1.2. 1 H NMR urinalysis The urine was collected in polypropylene tubes, promptly frozen in liquid nitrogen, and stored at 2 308C until used. A 50-ml volume of 2 H 2 0 was added to 0.5 ml of urine samples for field / frequency locking. Sodium tetradeutero-3trimethyl-silylpropionate (TSP), 5.8 mmol / l, was dissolved in the 2 H 2 0 to serve
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
119
as a reference for the chemical shifts and peak areas. NMR measurements were made on an FT-NMR spectrometer (Jeol EX270, Jeol, Tokyo, Japan) operating at 270.05 MHz in the homogated decoupling mode. All spectra were recorded at 258C. The spectral conditions were: spectral width 5405.4 Hz, acquisition time 3.28 s, pulse width 5.0 ms, delay 5.00 s, and transient 40. The signal for H 2 0 near 4.8 ppm was reduced by the presaturation method. The signal assignments were referred to the chemical shifts of metabolites previously reported [3–6]. Urinary concentrations of alanine, ketone bodies (3-hydroxybutyrate, acetone and acetoacetate) and lactate were estimated from the ratios of their peak areas to those of TSP. Relative concentrations of glucose were obtained as the relative peak heights at 3.7 ppm to those of TSP because of their multiple peaks. The urinary excretion rates of these metabolites were calculated thus: urinary excretion rate of the metabolites (mmol ? kg 21 ? h 21 ) 5 urinary concentration of the metabolites (mmol ? l 21 ) urine output per hour (l ? h 21 ) / body weight (kg)
2. Results Patient characteristics are shown in Table 1. Three of the seven patients underwent gastrectomy, two underwent mastectomy, and two underwent minor surgery. All patients recovered from anesthesia without apparent metabolic complications such as consciousness disturbances.
2.1. Signal assignments Fig. 1 shows a typical 1 H NMR spectrum of urine from a healthy volunteer. The spectrum was complex, with many signals being detected within the few minutes of total scanning time. A wide range of urinary metabolites could be identified in the chemical shift range of 1–4.2 ppm which is consistent with previous reports [3–5], but many minor resonances from metabolites in low Table 1 Characteristics of the patients Case a
Age (years) / gender
Operation
Operative time (h)
G1 G2 G3 M1 M2 T L
52 / M 49 / M 60 / F 60 / F 56 / F 29 / M 22 / M
Total gastrectomy Total gastrectomy Total gastrectomy Radical mastectomy Radical mastectomy Construction of tympanum Repair of cleft lip
5 4.5 3.5 4 4 5 4
a
Initials represent the operation.
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T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
Fig. 1. A typical 1 H NMR spectrum of urine from a healthy volunteer (26 year old male). Ala, alanine; Bu, 3-hydroxybutyrate; Ch, choline; Cit, citrate; Cn, creatinine; DMA, dimethylamine; Glu, glucose; Gly, glycine; Lac, lactate; N-Ac, N-acetylated glycoprotein; TMAO, trimethylamineN-oxide.
concentrations remain to be assigned. The strongest signals, at 3.05 and 4.08 ppm, were from creatinine, and the peaks at 3.23, 3.29 and 3.57 ppm were assignable to choline, trimethylamine-N-oxide, and glycine, respectively. Glucose showed multiple peaks in the relatively crowded region of the spectrum between 3.2 and 4.0 ppm. The small double peaks at 1.25, 1.33, and 1.47 ppm have been assigned to 3-hydroxybutyrate, lactate, and alanine, respectively. N-Acetylated glycoprotein gave rise to a single peak at 2.04 ppm. Double peaks around 2.6 ppm were assigned to citrate, and dimethylamine showed a single peak at 2.73 ppm.
2.2. Serial 1 H NMR spectra and urinary excretion rates of metabolites Fig. 2 shows the time course of the 1 H NMR spectra of urine collected in case G2. The 1 H NMR spectra did not change significantly during the first hour after the incision, but the 3-hydroxybutyrate and acetone signals appeared after the end of the first hour. The most remarkable changes were that the signal intensities of lactate and glucose significantly increased 2 h after the incision and throughout the rest of the surgery. The creatinine signals did not show significant changes at any time during surgery. The time-course changes in the urinary excretion rates of metabolites in each
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
121
Fig. 2. Time-course of the 1 H NMR spectra of urine from case G2. Time is shown in hours post-incision. Urine outputs per hour were 200, 50, 70, 60, 50, 60 ml (from the top). An, acetone; Bu, 3-hydroxybutyrate; Cn, creatinine; Glu, glucose; Lac, lactate.
patient are summarized in Fig. 3. The rates of alanine excretion significantly increased over 2–3 h in cases G2, G3, and M2. The 3-hydroxybutyrate and acetone excretion rates appeared to increase during the latter half of surgery in
122
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
Fig. 3. Time-course changes in the urinary excretion rates of metabolites in each patient. * Relative value.
cases G1, G2, G3, M1, and T. Acetoacetate showed significant increases over 1–2 h in cases G2, G3, and M1, and at 4–5 h in case G1. Lactate significantly increased at 2–3 h in cases G2 and M2, and further increased throughout the remainder of surgery in case G2. Cases G2 and M2 showed marked increases in glucose at 3–4 and 2–3 h, respectively. Case L did not show significant changes in the excretion rates of metabolites at any time during surgery except small increases of glucose at 0–1 and 1–2 h.
3. Discussion The NMR spectra of urine from most of the intraoperative patients showed remarkable increases in urinary excretion of alanine, ketone bodies, and lactate, and / or glucose. These metabolic profiles confirm the energy substrate–endocrine alterations that occur during surgery [1]. The increased excretion of alanine may be explained by accelerated protein catabolism for gluconeogenesis induced by surgical trauma. The appearance of ketone bodies is probably associated with the release of fatty acids as a fuel source. Glycogenolysis in muscle and / or decreased tissue perfusion may explain the increased lactate excretion. The increased glucose excretion may be caused by increased hepatic glucose output and / or the reduced peripheral use of glucose known as ‘diabetes of trauma’. These metabolic changes appeared to be roughly proportional to the degree of surgical stress, since patients T and L, who underwent minor surgery, showed
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
123
small changes in the urinary excretion of the metabolites. However, they were not consistent among the patients who underwent same operation: case G2 showed increased excretion of all urinary metabolites, while the rates of excretion of alanine and ketone bodies increased in case G3, and ketone bodies alone increased in case G1. It is also noteworthy that these metabolic changes became prominent 2 or 3 h after the incision was made, a finding consistent with the results of our previous study on serum metabolites [7]. The increased excretion of urinary metabolites during surgery tended to return to preoperative levels at the end of surgery, but in some cases it remained increased or rather enhanced at the end of surgery. The time course of 1 H NMR spectra thus allows close monitoring of the pathological status of patients during all phases of surgery and recovery. In addition, these findings demonstrate that intraoperative metabolic alterations are difficult to anticipate, and therefore routine intraoperative monitoring of cellular metabolites especially their time course is necessary to obviate critical metabolic disorders. The current study demonstrates two unique advantages of 1 H NMR spectroscopy of urine for perioperative metabolic monitoring. First, it allows rapid multicomponent analysis of urinary metabolites. The detection of a wide range of metabolites is of particular clinical usefulness when unchecked metabolic alterations due to preoperative starvation and surgical trauma have the potential to develop into critical metabolic derangements. In addition, almost all real-time metabolic monitoring is attractive, because surgeons are sometimes obliged to change the scheduled operative procedure to a simpler and less stressful one in the face of life-threatening metabolic disorders such as severe ketosis, hyperglycemia, and / or lactic acidosis. Another advantage is that the NMR spectra patterns of energy substrates, such as amino acids, ketone bodies, lactate, and glucose provide information on perioperative fuel metabolism characterized as the catabolic state [1]. For example, the NMR spectrum patterns of urinary amino acids as well as glucose may be helpful in deciding the optimal intraoperative dose of glucose as a fuel source, which is supposed to suppress protein catabolism [1]. In the routine application of 1 H NMR spectroscopy of urine for cellular metabolic monitoring, some problems must be resolved. 1 H NMR spectroscopy is a relatively insensitive method capable of detecting species at around 0.1 mmol / l, with precise quantification being uncommon because of signal line broadening or overlapping [3,4]. Nevertheless, once metabolites showing abnormal excretion profiles are detected in the wide screen of the 1 H NMR spectrum, appropriate evaluation and therapy for this abnormality can be instituted, and more sensitive analysis then can be readily performed saving cost and time for unnecessary specific tests. It is true that the high-field NMR instruments are expensive, but their convenience and cost effectiveness is also
124
T. Tatara et al. / Clinica Chimica Acta 279 (1999) 117 – 124
valuable because their maintenance costs are low and instruments can process samples around the clock in an automated manner [4]. The NMR spectra should be carefully analyzed considering confounding variables such as drugs which may influence NMR spectra. This problem may be resolved by obtaining the NMR signals of drugs alone and measuring time course of the NMR spectra from the administration of the drugs. The variations of the NMR spectra within normals are unlikely to become a serious confounding factor as long as the time course of the NMR spectra is discussed. Although the utility of 1 H NMR spectroscopy of urine for metabolic monitoring has not been widely appreciated by clinicians, our results suggest that 1 H NMR spectroscopy of urine, especially the time-course findings, provide a novel approach to cellular metabolic monitoring during surgery.
References [1] Biebuyck JF, Stene JK. Nutritional aspects. In: Miller RD, editor. Anesthesia, 3rd ed. New York: Churchill Livingstone, 1990:2277–2305. [2] Tonnesen AS. Monitoring the kidney and urine. In: Blitt CD, editor. Monitoring in Anesthesia and Critical Care Medicine, 2nd ed. New York: Churchill Livingstone, 1990:575– 594. [3] Nicholson JK, Wilson I. High resolution proton magnetic resonance spectroscopy of biological fluids. Prog NMR Spectrosc 1989;21:449–501. [4] Reglinski J, Watson ID. An analytical perspective of NMR spectroscopy of biological fluids and cells. Ann Clin Biochem 1996;33:290–307. [5] Foxall PJD, Mellotte GJ, Bending MR, Lindon JC, Nicholson JK. NMR spectroscopy as a novel approach to the monitoring of renal transplant function. Kidney Int 1993;43:234–45. [6] Sweatman BC, Farrant RD, Holmes E, Ghauri FY, Nicholson JK, Lindon JC. 600 MHz 1 H-NMR spectroscopy of human cerebrospinal fluid: effects of sample manipulation and assignment of resonances. J Pharm Biomed Anal 1993;11:651–64. [7] Tatara T, Noguchi J, Fukushima K, Ishihara Y, Ohkouchi S, Uedaira H. Proton NMR spectroscopic studies of serum as an aid to perioperative cellular metabolic monitoring. Physiol Chem Phys Med NMR 1995;27:121–9.