Interrelation between whole-body turnover rates of RNA and protein.

In our search for new non-invasive methods to determine metabolic and nutritional state, we have identified several specific, modified, urinary one-way catabolites of rRNA, tRNA and mRNA which permit the assessment of the whole-body turnover of these RNA classes. A comparison of the steady-state turnover of RNA and the proteins actin plus myosin (determined using urinary 3-methylhistidine) in preterm infants and adults showed that preterm infants have about 3 times higher average turnover rates per unit body weight than adults of tRNA and rRNA as well as of actin plus myosin, whereas calculated mRNA turnover was 6 times higher in preterm infants than in adults. These as well as our recent observations of RNA turnover in different mammals are compared here with data on whole-body protein turnover and basal metabolic rates (BMR) in different mammalian species including man, for which data are available. The turnover rates of tRNA, rRNA, protein and energy (BMR) can be described by the relation, turnover = const. x body mass (exp.), the extrapolated exponents being 0.69-0.78. This suggests a common underlying principle, possibly energy turnover, as cause for the coordinated whole-body turnover rates of RNA and protein in the steady state.

Determination of 7-methylguanine, N2,N2-dimethylguanosine, and pseudouridine in ultrafiltrated serum of healthy adults by high-performance liquid chromatography.

7-Methylguanine (m7Gua), N2,N2-dimethylguanosine (m2(2)Guo), and pseudouridine (psi) are degradation products from RNA turnover and can be used as markers for the whole-body turnover of mRNA-cap, tRNA, and rRNA (in healthy individuals, urinary excretion of these catabolites follows a regular pattern; the relative molar ratio of psi:m7Gua:m2(2)Guo is approximately 100:19:6). HPLC methods were developed to measure serum concentrations of these RNA catabolites after deproteinization of the samples by ultrafiltration through microcollodion bags with a nominal exclusion Mr of 12,400. For healthy adults the following values (mean +/- SD) were found: psi, 2760 +/- 460 nmol/liter (n = 10); m7Gua, 129.7 +/- 24.0 nmol/liter (n = 13); m2(2)Guo, 31.0 +/- 3.7 nmol/liter (n = 9). The relative molar ratio of these substances in serum derived from our data is approximately 100:4.7:1.1. 7-Methylguanosine (m7Guo) added to serum is to a large extent converted to the corresponding free base, m7Gua, the form which is excreted in urine.

Ribonucleic acid turnover in man:RNA catabolites in urine as measure for the metabolism of each of the three major species of RNA.

Urinary excretion of the non-reusable modified RNA catabolites pseudouridine (psi), 7-methylguanine (m7Gua) and N2,N2-dimethylguanosine (m2(2)Guo) was measured in preterm infants and in adults. The values (in mumol/mmol of creatinine) were: for preterm and 'small for gestational age' infants (n = 26; number of samples = 38) psi = 164 (SD 32), m7Gua = 39.1 (SD 9.0), m2(2)Guo = 10.6 (SD 2.1); for adults (n = 32) psi = 25.3 (SD 3.1), m7Gua = 4.8 (SD 0.89), m2(2)Guo = 1.53 (SD 0.42). Our measurements were compared with an expectation derived from the average cellular distribution of psi, m7Gua and m2(2)Guo between rRNA, tRNA and mRNA. m2(2)Guo occurs exclusively in tRNA, psi in both rRNA and tRNA, and m7Gua in all three RNA classes, in proportions which can be estimated for the steady state. Urinary excretion of psi and m2(2)Guo should reflect their steady-state distribution, since rRNA and tRNA have been shown to have similar turnover rates in mammalian tissues. We conclude that we can use the excretion of m2(2)Guo to assess whole-body tRNA turnover. Since tRNA contains psi in a constant proportion to m2(2)Guo, the proportion of urinary psi stemming from tRNA can be estimated, and the remainder (approximately 60-65%) is an indicator of rRNA turnover. Finally, the excretion of m7Gua far exceeds the proportion predicted to come from rRNA and tRNA. We ascribe this excess (approximately 60-70% of the total) to the turnover of the mRNA 'cap'-structure, which is typical for all higher organisms. mRNA turnover is known to be much higher than that or rRNA or tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible use of urinary modified RNA metabolites in the measurement of RNA turnover in the human body.

Modified building blocks are found in rRNA, tRNA and mRNA. Apart from pseudouridine these are mostly base- or ribose-methylated nucleosides. If these compounds are neither recycled nor degraded, they should be quantitatively excreted. For pseudouridine (Weissman et al., 1962; Dugaiczyk & Eiler, 1966) and 7-methylguanine (Craddock, Mattocks & Magee, 1968), urinary excretion has been shown to be quantitative. Since the turnover rates of rRNA and tRNA, which contain most of the modified nucleosides, are similar within a given tissue, compounds found only in these two classes of RNA should appear in urine in approximately the proportions in which they are present in the body. Using pseudouridine as internal standard, we show this indeed to be likely for one of the major RNA catabolites in human urine, N2,N2-dimethylguanosine, a compound present only in tRNA. By contrast, 7-methylguanine is excreted in threefold larger amounts than can be explained by joint provenance from tRNA and rRNA only; the remainder we assume to come from the 'cap' structure of mRNA, known for its high turnover. We suggest that one can use the urinary excretion of pseudouridine, N2,N2-dimethylguan(os)ine and 7-methylguanine to assess the whole-body turnover rates in man of rRNA, tRNA and mRNA, respectively. Such data may be useful to define whole-body metabolic activity.

Protein and RNA turnover in preterm infants and adults: a comparison based on urinary excretion of 3-methylhistidine and of modified one-way RNA catabolites.

Urinary excretion of 3-methylhistidine in preterm infants (n = 42; 1,712 +/- 408 g, 4-91 days old) was 24.2 +/- 6 mumol/mmol creatinine or 2.26 +/- 0.56 mumol/kg body weight X day. In adults (n = 6; 66 +/- 10 kg, 17-50 years), the corresponding values were 10.5 +/- 1.1 mumol/mmol creatinine and 2.21 +/- 0.23 mumol/kg body weight X day. For both collectives, the breakdown per kg body weight of 3-methylhistidine-containing protein (i.e. actin and myosin) was similar, at approximately 0.7 g/kg X day (preterm infants 0.84, adults 0.60). Since the preterm infants studied contain approximately 21% muscle instead of the 43% found in adults, the 3-methylhistidine excretion in preterm infants probably indicates muscle (and intestinal) protein turnover to be about 3 times higher than in adults, a figure in accord with data on whole-body protein turnover in preterm infants and adults (approximately 15 g/kg X day and approximately 4 g/kg X day, respectively). Urinary excretion of pseudouridine (psi), 7-methylguanine (m7Gua) and N2, N2-dimethylguanosine (m2(2)G) can be used to estimate the turnover of rRNA, mRNA and tRNA, respectively. The values obtained (in mumol/mmol creatinine) in preterm infants are for psi: 164 +/- 32; for m7Gua: 39.1 +/- 9; and for m2(2)G: 10.6 +/- 2.1. In adults, the values are for psi: 25.3 +/- 3.1; for m7Gua: 4.8 +/- 0.89; and for m2(2)G: 1.53 +/- 0.38. This yields 3-4 times higher turnover rates in preterm infants than in adults for all 3 RNA classes: rRNA, 0.1 versus 0.038; tRNA, 1.87 versus 0.66; mRNA 2.35 versus 0.64 mumol/kg X day.

A high-performance liquid chromatographic method for the determination of pseudouridine and uric acid in native human urine and ultrafiltered serum.

A simple and reliable HPLC method for quantitative determination of pseudouridine and uric acid in human urine and serum using a cation-exchange resin is described. This method is straightforward (12 runs of urine samples per day since the sample is only diluted into buffer and then chromatographed), sensitive, and highly reproducible. The column is stable over long periods (approximately 3 months of uninterrupted use at a time; it is thereafter easily restored to the original state). Mean excretion values for pseudouridine (in mumol/mmol creatinine) are 26.4 +/- 3.1 (17 female adults), 23.8 +/- 2.5 (12 male adults), 164.7 +/- 32.2 (37 male preterm infants); mean values for uric acid (mumol/mmol creatinine) are, respectively, 310.3 +/- 90.5, 278.2 +/- 56.1, and 1108 +/- 314. Human serum is deproteinized by pressure ultrafiltration in microcollodion bags with a nominal exclusion molecular weight of 12,400 and then put directly onto the HPLC column. The complete procedure takes 4 h.

[Excretion of normal and modified RNA catabolites and creatinine in the urine as a function of nutrition in children]. / Die Ausscheidung normaler und modifizierter RNA-Kataboliten sowie von Kreatinin im Urin in Abhängigkeit von der Ernährung bei Kindern.

The urinary excretion of 5 modified and 2 unmodified RNA catabolites and of creatinine has been investigated in two children aged 71/2 and 6 years under different isocaloric diets over 5 days each (phase I-III) and compared with the excretion values during a 12-day and libitum food intake (phase 0). On a diet (phase I) extremely rich in nucleic acids (meat) there is only a slight increase of the modified RNA catabolites and a pronounced increase of creatinine in urine. On a protein-rich diet free from nucleic acids (phase II) the urinary RNA catabolites largely parallel the values seen under ad libitum food intake. On a diet virtually free of nucleic acids and protein (phase III) there is a markedly decreased excretion of modified RNA catabolites. It is concluded that modified RNA catabolites in the urine are mainly of endogenous origin. These "one-way catabolites" permit an assessment of the RNA turnover. Thus, they can serve as a new sensitive biochemical criterion for anabolic and catabolic situations, respectively.

Multivariate analysis of urinary RNA catabolites in malignancies: cross-sectional and longitudinal studies.

Urinary RNA catabolites, especially modified nucleosides and nucleobases, have turned out to represent valuable new criteria for diagnosis and follow-up of malignancies. Here we show for the first time that multivariate analysis of urinary RNA catabolites can distinguish between tumor carriers and controls who, if examined by univariate procedures, would remain undifferentiated. Two such models have been demonstrated. We hope that a continuation of this work will support primary clinical diagnostic procedures, the control of therapy effects, and the long-term follow-up of patients, which should be regarded as special medical applications of the general principles of molecular biology.

[Age dependency of normal and modified nucleobases in urine in correlation to growth velocity (author's transl)]. / Die Altersabhängigkeit der normalen und modifizierten Nucleobasen im Urin als Ausdruck der Wachstumsgeschwindigkeit.

Up to now no age-correlated normal values of normal and modified nucleobases in urine have been published. In this paper such normal values are presented, obtained from 116 healthy subjects from 0 to 16 years by high performance liquid chromatography of creatinine-correlated single urine specimens. A narrow correlation between the excretion of nucleobases and growth velocity was found. A polynomial function including the puberal growth spurt could be fitted to the analytical data. It is felt that this method should be suitable to assess growth processes of different kinds, e.g. effects of nutrition or drugs and hormones on growth velocity, furthermore intrauterine and malignant growth.

[Normal and modified nucleobases excreted in urine of patients with chronic nyeloproliferative disorders (author's transl)]. / Die Ausscheidung von normalen und modifizierten Nucleobasen im Urin bein chronischen myeloproliferativen Syndromen.

The qualitative and quantitative analysis by high performance liquid chromatography of the normal and modiefied nucleobases excreted in urine represents a new and versatile tool, especially in oncology. The excretion of 2 normal (adenine, guanine) and 4 modified nucleobases (methylated guanine derivatives) in urine was measured by cation exchange LC. All chronic myeloproliferative syndromes showed highly elevated total excretion values of all determined nucleobases, the "pattern" being characteristic with N2, N2-dimethylguanine most prominent (up to 29.8 S.D. above the pertinent normal value). A follow-up study of a case of CML with two episodes of extreme leukocytosis showed a correlation of the nucleobases excretion with the number of leukocytes. Thus, a method has been established which permits the assessment of myeloproliferation and probably therapy effects.

[Analysis of normal and methylated urinary nucleobases: a new criterion for diagnosis and follow-up of malignancies (author's transl)]. / Die Analyse von normalen und methylierten Nucleobasen im Urin als neues Kriterium für Diagnose und Verlauf von Malignomen.

The qualitative and quantitative analysis by high performance liquid chromatography of the normal and modified nucleobases excreted in urine represents a new and versatile tool. Its use for the diagnosis of malignancies, deliminating infectious diseases, and for the follow-up of the effects of a cytostatic therapy is shown. This method offers a chance to introduce the analysis of a series of fragments of primary gene products into the routine of clinical biochemistry.