Application of proton NMR spectroscopy to measurement of whole-body RNA degradation rates: effects of surgical stress in human patients

ELSEVIER

Clinica Chimica Acta 252 (1996) 123-135

Application of proton NMR spectroscopy to measurement of whole-body RNA degradation rates: effects of surgical stress in human patients Jaspaul S. Marway *a, Graeme J. Anderson b, John P. Miell c, Richard Ross c, George K. Grimble a, Adrian B. Bonner a, William A. Gibbons d, Timothy J. Peters e, Victor R. Preedy e aTissue Pathology Unit, Roehampton Institute London, West Hill, London, SW15 3SN. UK bDepartment of Chemistry, Manchester Metropolitan University, Chester Street, Manchester MI 5GD, UK CDepartment of Medicine, King's College School of Medicine and Dentistry, Bessemer Road, London, SE5 9PJ, UK aDepartment of Pharmaceutical Chemistry, School of Pharmacy, London, UK CDepartrnent of Clinical Biochemistry, King's College School of Medicine and Dentistry, Bessemer Road, London, SE5 9PJ, UK

Received 9 January 1996; accepted 29 January 1996

Abstract

The urinary catabolites, N2,N2-dimethylguanosine (DMG), pseudouridine (PSU) and 7-methylguanine (mT-Gua) are formed from post-transcriptional methylation of RNA bases and are not reincorporated into RNA upon its degradation. Their quantitative urinary excretion may be used to determine rates of whole body degradation of individual RNA species since DMG occurs exclusively in tRNA, PSU occurs in rRNA and tRNA and mT-Gua occurs in all RNA species. Conventional HPLC analysis has several drawbacks since pre-analytical steps may involve selective losses and, under certain conditions, other urinary analytes may co-elute. In the present paper, we report analysis of these compounds by high-field 1H-nuclear magnetic resonance (1H-NMR) spectroscopy. Urinary concentrations of these metabolites were found to be in agreement with previously published HPLC and ELISA determinations. However, NMR analysis required minimal sample preparation (other than lyophilisation and reconstitution) and was capable of the simultaneous AbbrevJ!ation.w DMG, N2,NZ-dimethylguanosine; PSU, pseudouridine; m~-Gua, 7-methylguanine.

*Corre:~ponding author, Tel: +44-181-3923549; Fax: +44-181-3923691. 0098-8981/96/$15.00 ~, 1996 Elsevier Science B.V. All rights reserved SSDI S0009-8981(96)06300-Q

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determination of other relevant analytes such as creatinine. This technique was therefore applied to urine samples from patients who had undergone surgical stress and insulin-like growth factor-I (IGF-1) therapy. Surgical stress increased the excretion of DMG and mT-Gua. Degradation rates for tRNA and mRNA were also higher in surgically stressed subjects when compared with controls but degradation rates of rRNA decreased by approx. 30%. However, injection of IGF-I (40 pg/kg s.c.) had no significant effect on the excretion of these nucleosides. These data indicated that IGF-I therapy has no marked effects on RNA turnover following trauma. We suggest that this technique can be applied to study of RNA metabolism in any surgical or medical condition. Furthermore, since only 0.6 ml of urine is required, studies in neonates seem .to be feasible.

Keywords: Proton nuclear magnetic resonance; Urine; RNA catabolites; IGF-I; Surgical stress

1. Introduction

Catabolic conditions resulting in whole-body nitrogen loss occur in several pathological states, including malnutrition, cardiac cachexia, endocrine abnormalities and alcoholism [1]. These losses may occur as a result of changes in either protein synthesis and/or degradation that can occur secondary to alterations in the amount of protein synthesising machinery which can be represented by tissue RNA concentration [1-3]. We have shown that there is a direct correlation between the number of ribosomes in intestinal tissue and its rate of protein synthesis in vivo [4], as has been reported for skeletal muscle [5]. These data would suggest that more attention be paid to the regulation of RNA content and especially the turnover rates of individual RNA species. In addition to the major nucleosides (adenosine, guanosine, uridine and cytidine), all three major RNA species contain components formed by post-transcriptional modification. Most of these modified components cannot be reutilised and are excreted in urine [6]. In human urine more than 40 nucleosides have been identified, among which N2,N2-dimethylguanosine, pseudouridine and 7-methylguanine occur at comparatively high levels. Their excretion is quantitative [7-9]. Therefore, excretion of these modified nucleosides in urine has been studied with respect to their biomedical significance as possible markers of cancer [7,10] as well as AIDS [11]. As the content of these modified RNA catabolites in their parent macromolecule (mRNA, rRNA and tRNA) is known, their urinary excretion provides a suitable marker for the determination of degradation rates of mRNA, rRNA and tRNA [8-9]. This is analogous to the use of urinary 3-methylhistidine excretion as an index of myofibrillar protein breakdown (for example [12]).

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Although HPLC and ELISA techniques for the assay of the RNA nucleotides existed, they were time-consuming (particularly HPLC); not all nucleotides could be measured in the same assay (particularly ELISA methods); some lengthy preparative steps were inefficient and resulted in losses and some nucleotides were not resolved adequately in the H P L C assay systems. In contrast the application of high field IH-NMR spectroscopy to the study of biological fluids from various patient groups may circumvent these problems. As has been shown by several studies (e.g. 1-13-17]) a wide range of metabolites may be detected and quantified in various body fluids as well as tissue extracts using this technique which is capable of simultaneous analysis of many analytes in the same sample. In the present study we have therefore applied this technique to urine analysis in critically ill patients who had undergone major abdominal surgery. In recent studies [18] a consistent finding in these patients was that circulating levels of the anabolic agent, insulin-like growth factor-I (IGF-I) were reduced whilst blood growth hormone (GH) concentrations were increased. This suggested that GH resistance had developed and as IGF-I mediates many actions of GH, the hypothesis under test was whether recombinant IGF-I therapy could reverse the catabolic state in these patients. 1H-NMR of urine was therefore used to assess whole body RNA degradation rates. 2. Materials and methods 2.1. Materials N2,N2-Dimethylguanosine, pseudouridine and 7-methylguanine were purchased from Sigma Chemical Company (Poole, Dorset, UK). Deuterium oxide (D20) was purchased from Merck Frost Canada Inc. (Cambrian Gases, Croydon, UK). Other materials were from various suppliers and were of the highest grade available. 2.2. Urine specimens For evaluation of N M R as a tool for determination of concentrations of RNA catabolites, spot specimens of urine were collected from healthy male persons without any known metabolic diseases. On collection of urine, aliquots (typically 2.5 or 4 ml) were frozen at -20°C. All subjects were males aged between 29 and 45 years. To investigate the effects of surgical stress and IGF-I therapy, 24-h urine samples from a group of 30 patients aged between 45 and 75 years undergoing major surgery were collected. Sample collection commenced at 08:00 h on the day following surgery. On the second post-operative day, patients received a single subcutaneous injection of either recombinant

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IGF-I or placebo. Urine specimens (24 h) were collected 96 h following surgery (48 h post IGF-I injection) and assayed for RNA catabolites. Other clinical aspects of this study have been published previously 1-19].

2.3. Preparation of urine samples for proton N M R spectroscopy Aliquots of urine (2 ml) were lyophilised and reconstituted in 1.2 ml of phosphate buffered deuterium oxide (10 mmol/1; pH 7.0) containing a known concentration of sodium 3-(trimethylsilyl)-l-propionate (TSP; 6 = 0 ppm). The pH was checked, corrected to pH 7 with N a O D or DC1 and samples were filtered through a 0.22-/~m Millipore filter. Aliquots of 0.6 ml were transferred to a 5 m m N M R glass tube and the spectra recorded.

2.4. Proton NMR spectroscopy Proton N M R spectra were recorded with a Bruker AM500 N M R spectrometer (11.7 T) operating at 500.14 MHz. The 1D proton spectra were recorded at 293 K in the Fourier transform mode. A 60 ° pulse was applied with a spectral width of 5000 Hz and 1024 or 2048 free induction decays (FIDs) were collected with 16 384 data points prior to transformation. During a relaxation delay of 4 s the H z O / H D O signal at about 4.7 p p m was suppressed by homogated decoupling. All peaks were referenced to the standard (TSP) at 0.00 ppm.

2.5. Identification of resonances (peak assignment) and determination of concentrations The concentrations of metabolites in urine were determined by comparison of the identified resonance peak areas with that of a known amount of TSP. In all calculations the number of protons were taken into consideration. To confirm the assignments, urine samples were spiked with pure standard. To assess analytical recovery, known concentrations of D M G , PSU and mV-Gua in phosphate buffered DzO were added to urine samples and assayed. In addition, to determine the effects of sample concentration, various volumes of urine were freeze-dried and re-dissolved in 0.6 ml phosphate buffered DzO before analysis.

2.6. Statistics All data are presented as means ___S.E.M. of 3 - 7 observations. In the study of the effects of surgical stress and IGF-I treatment on whole-body RNA degradation, all data are presented as means ___S.E.M. of 9-20 observations. Differences between means were tested by ANOVA.

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3. Results

The complex proton resonance signals derived from normal urine are illustrated by the spectra presented in Fig. 1. However, the spectral assignments of the three main RNA catabolites were tentatively obtained from the known structures of the metabolites and confirmed by "spiking" samples with pure standards. The unique chemical shifts of N2,N 2dimethylguanosine (DMG; 3.16 ppm), pseudouridine (PSU; 7.69 ppm) and 7.-methylguanine (mT-Gua; 7.88 ppm) are shown in Fig. 2. The: data presented in Table 1 shows the concentrations of these three main RNA catabolites excreted in urine as determined by NMR. These were in good agreement with previously published data obtained by ELISA or H P L C (Table 1). The percentage recovery of N2,N2-dimethylguanosine, pseudouridine and 7-methylguanine, were found to be 92 _ 5%, 94 _ 2% and 91 _ 2%, respectively (all data means + S.E.M. of 3-4 observations). In addition to determining the analytical recovery,

do

do

do

ppm Fig. 1. Complete 1-dimensionalproton NMR spectra (500 MHz) of urine from normal adult.

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PSU m7-Gua

/o /.31

s:oo

7.'95 r.'9o

7'as

I

r'.o

7.~s

r.'7o

r'es

PPM

nMG e

3'.25

3.20

3.15

3.'10

3.05

PPM

Fig. 2. One-dimensional proton NMR spectra of urine from normal adult showing modified nucleosides in the region of higher field (top) and that of lower field than the H20/HDO signal (bottom). DMG, N2,N2-dimethylguanosine; PSU, pseudouridine; m 7Gua, 7-methylguanine.

we also investigated the effects of concentrating urine on relative amounts of RNA catabolites. As shown in Fig. 3 there was a linear relationship between the concentration of RNA catabolites, determined by NMR, and the volume of urine freeze dried and reconstituted with a fixed volume (0.6 ml) of buffered D 2 0 .

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Table 1 Comparison of IH-NMR, HPLC and ELISA methods for analysis of urinary RNA catabolites n

DMG

PSU

mT-Gua

Reference

Urinary excretion (/~mol/mmol creatinine) tH-NMR. Spot urine samples collected over 3 month period. Single male subject. tH-NMR. Spot urine samples. Male subjects HPLC ELISA

6

2.6 -t- 0.2

26.7 + 1.6

3.5 -I- 0.2

This study

7

2.1 -I- 0.4

33.8 __+ 1.9

3.1 _ 0.6

This study

1.5-2.2 --

20.3-31.2 31.2 4- 9.9

3.5-4.8 --

[8,9,30,311 [32]

32-74 33

The concentration of urinary RNA catabolites were determined by =H-NMR in random "spot" urine samples from a single male subject over a 3 month period and in urine samples from seven healthy male subjects aged between 29 and 41 years. All data are means 4- S.E.M. of 6 - 7 observations. DMG, NZ,NZ-dimethylguanosine; PSU, pseudouridine; mV-Gua, 7-methyiguanine.

Table 2 shows urinary excretion concentrations of RNA catabolites (expressed relative to creatinine) in urine from control, surgically treated and IGF-I or placebo treatment groups. Table 2 clearly shows that with

~,

600

o Pseudouridine

-6

.o

40o

L~ 200 o (,q

0

"

0

1

I

I

I

I

2

3

4

5

Urine Volume (ml)

Fig. 3. Effect of urine volume on pseudouridine and 7-methylguanine levels by tH-NMR. Urinary RNA catabolite concentrations were determined by ' H - N M R using different concentrations of urine. All data are expressed as amount of RNA catabolite as = of value for 1 ml of urine. Each point represents the mean -I- S.E.M. of 3 - 4 urine samples from different subjects.

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Table 2 The effects of I G F - I a n d placebo o n R N A catabolites c o n c e n t r a t i o n s a n d t u r n o v e r rates for tRNA, rRNA and mRNA n

DMG

PSU

mT-Gua

Urinary excretion (#mol/mmol creatinine)

Controls Surgical stress IGF-1 Placebo

tRNA

rRNA

mRNA

Calculated average degradation rate (/~mol/kg per day)

7 19-20

2.14 + 0.37 3.60 + 0.42

33.8 ___1.9 35.6 + 3.0

3.11 ___0.57 8.43 + 2.25

0.88 1.48

0.0478 0.0334

0.105 0.848

10-11 9

3.78 +__0.9 4.14 ± 1.35

46.4 ± 4.0* # 6.29 ± 1.52 50.1 ± 4.0**+ + 6.67 ± 1.81

1.56 1.71

0.0549 0.0586

0.353 0.340

DMG, N2,N2-dimethylguanosine; PSU, pseudouridine; m%Gua, 7-methylguanine. *P < 0.05, **P < 0.025, control group; ~P < 0.05, ¢¢P < 0.01, versus surgical stressed. The concentration of urinary RNA catabolites were determined by IH-NMR in urine samples from patients undergoing major abdominal surgery (gastrectomy or colonic resection with anastomosis). The pre- and post-placebo groups represent urinary collections before and after placebo treatment, respectively, following surgery. RNA turnover data were calculated using formulae as described by Sander et al. [8]. Calculated turnover rates for tRNA, rRNA and mRNA in healthy adults as published previously: 0.63, 0.037 and 0.62 /~mol/day per kg body weight, respectively [8]. All data pertaining to urinary excretions are presented as means + S.E.M. of 9-20 observations. Although surgically stressed subjects were older, one should expect a decrease in RNA degradation rates; however, an increase was observed reflecting metabolic perturbation due to the disease process or trauma.

placebo treatment concentrations of D M G and PSU increased by 15% (P>0.05) and 41% (P < 0.01), respectively, whereas concentrations of mT-Gua decreased by 21% (P > 0.05). As shown in Table 2 with IGF-I treatment, urinary excretion of PSU increased by 30% (P < 0.05). The concentration of D M G was not effected but urinary mV-Gua concentration decreased by 25% (P > 0.05). Determination of RNA degradation rates showed that with placebo treatment rRNA and tRNA degradation rates increased by 75% and 15%, respectively, whereas mRNA degradation rates decreased by 60%. However, it should be noted that the urinary excretion of DMG, PSU and mV-Gua continued to increase to the fourth post-operative day because of continued catabolism. High tRNA and mRNA degradation rates were maintained in all surgically operated groups, except that rRNA degradation rates decreased by 30% 24 h after surgery when compared with controls. 4. Discussion

NMR is a powerful tool in chemistry and recently there has been an

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upsurge in its use in biochemistry and medicine. In the main, this has arisen from the construction of superconducting magnets with higher magnetic fields enabling better spectral resolution and thus allowing the analysis of complicated biological fluids. The technique is particularly attractive as it is non-invasive, requires very little sample and no complicated pre-extraction procedures (which can be time consuming and lead to substantial losses) are required before analysis by NMR. In this paper we present the assignments of the spectra for the three main RNA catabolites (Fig. 2) that are used for the determination of degradation rates of rRNA, tRNA and mRNA from human urine samples. In addition, we have shown that there was good analytical recovery of all the nucleosides added to human urine and that this was independent of the amount of urine which was lyophilised in 0.6 ml DzO (Fig. 3). Pseudouridine was the simplest RNA catabolite to quantify due to its relative abundance and its particular resonance signal in an uncrowded aromatic region of the spectra. The quantification of mT-Gua and D M G was relatively more difficult and required a larger number of scans and hence instrument time. In addition, to improve reproducibility, it was necessary to perform a manual baseline correction using identical baseline correction points for all spectra as opposed to an automatic baseline correction. As there was excellent agreement with previously published concentrations of the RNA catabolites in human urine, the present study confirms the utility of 1H-NMR for this purpose. We therefore applied this technique to a study of the effects of IGF-I administration to patients undergoing ' operative stress. Post-surgical trauma was of special interest due to the established well known effects surgery has on protein metabolism. Surgical trauma has long ago been shown to lead to loss of muscle protein [20]. As skeletal muscle constitutes the largest mass of protein in the body (about 40% of the total body weight) protein catabolism can have major effects on outcome and recovery. In this study net protein catabolism induced by surgical trauma may have been due to changes in protein synthesis and/or protein degradation. Certainly the increase in release of free amino acids from leg muscle following abdominal surgery indicates net protein catabolism [21]. Muscle protein synthesis has also been shown to decrease 24 h post-operatively while protein degradation, as indicated by urinary 3-methylhistidine excretion, has been shown to decrease [22,23]. In surgical trauma, factors such as stress hormone and cytokine release may evoke changes in protein synthesis. However, changes in protein synthesis or protein mass also suggest perturbations in RNA content or turnover rates. Our hypothesis was that in catabolic patients the turnover rates of some or all of the three

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main RNA species would be increased and that determination of RNA turnover could be used as a measure of catabolism. We also took the opportunity to study the effect of a possible therapeutic intervention (IGF-I) on RNA turnover. Whole-body protein metabolism is sensitive to stress, infection, injury and nutritional depletion. In addition, in many disease states (e.g. cancer cachexia and AIDS), weight loss is a characteristic feature of the disease process [24] and in many cancer patients the loss in weight is associated with reductions in skeletal muscle protein synthesis [25]. Recent studies on urinary excretion of modified nucleosides have identified changes in RNA turnover in some of the conditions. Specifically, RNA catabolite excretion and whole-body turnover rates of the three main RNA species have been found to be elevated in various forms of cancer and in patients with AIDS [11,26,27]. Most of the previous studies on RNA turnover have essentially investigated whole-body events rather than tissue specific events. In cancer patients elevated levels of PSU have been ascribed to a high turnover rates of tRNA in tumour tissue [28]. In the current study, the results for surgical stress showed that the turnover rates of tRNA and mRNA increased by approx. 2- and 8-fold, respectively, compared with the control group, but rRNA degradation rates decreased by 30%. With IGF-I treatment there appeared to be an increase in degradation of rRNA and conversely a reduction in m R N A turnover. In this study the urinary excretion of the RNA catabolites may be complicated by the effects of surgical stress induced by major abdominal surgery as well as by the large bowel cancer that the patients were operated for in the first place. Previous studies have shown that the excretion of urinary catabolites is elevated and shows variability in patients with large bowel malignancy [7] and in most cases the excretion of RNA catabolites is returned to normal levels after surgical removal of the tumour [29] or chemotherapy [7]. In our clinical study, tRNA and m R N A turnover rates in subjects on the first postoperative day before placebo or IGF-I treatment (i.e. surgically stressed) were higher than controls, but rRNA turnover rates were lower than in normal healthy adults (Table 2). One possible explanation for the high tRNA degradation rates could be that changes in protein turnover and a reduction in amino-acyl tRNA charging may make tRNA more susceptible to degradation. At 96 h following surgery in placebo or IGF-I treatment groups, high tRNA turnover rates are maintained compared with controls, although mRNA turnover rates decrease in the postoperative group given IGF-I or placebo when compared with the surgically stressed group. We can speculate that in cancer a high turnover rate of tRNA may be a characteristic feature of malignancy but surgical trauma may possibly be characterised by an increase in m R N A turnover.

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W e c o n c l u d e t h a t p r o t o n N M R is a valuable tool for identifying a n d q u a n t i f y i n g multiple c o m p o n e n t s in urine in a single o p e r a t i o n . This t e c h n i q u e has a distinct a d v a n t a g e o f offering the p o t e n t i a l of being able to easily identify a n d quantify multiple c o m p o n e n t s in urine s i m u l t a n e o u s ly w i t h o u t p e r f o r m i n g any c o m p l i c a t e d p r e t r e a t m e n t , e x t r a c t i o n or purification

Acknowledgements F u n d i n g f r o m the King's College R e s e a r c h S t r a t e g y F u n d is gratefully a c k n o w l e d g e d . J P M is a W e l c o m e Fellow. W e t h a n k Prof. E. S c h l i m m e a n d Dr. V. C r a d d o c k for the g e n e r o u s gift of N 2 , N 2 - d i m e t h y l g u a n o s i n e .

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