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Factors influencing the production of creatinine: Implications for the determination and interpretation of urinary creatinine and creatine in man
Clinica Chimicu Acta, 175 (1988) 199-210 Elsevier
199
CCA 04223
Factors influencing the production of creatinine: implications for the determination and interpretation of urinary creatinine and creatine in man Nigel J Fuller and Marinos MRC Dunn Clinical Nutrition (Received
Elia
Centre, Cambridge (UK)
20 October 1987; revision received 26 February accepted after revision 29 February 1988)
Key wordst Creatinine;
Creatine;
Creatine-phosphate;
Urine;
1988;
Equilibrium
Summary
The rates of creatine/creatinine inter-conversions and their equilibrium were studied under controlled conditions of temperature and pH that simulate urine storage conditions. The concentrations and ratios of creatine to creatinine in urine obtained from subjects with various pathophysiological conditions were determined, both before and after storage. The observed changes occurring during storage were compared with predicted changes based on observations of standard solutions. The initial reaction rate was found to increase with temperature, occurring maximally at about pH 3.7 for the conversion of creatine to creatinine, and at about pH 5.0 for the conversion of creatinine to creatine. At low pHs the equilibrium position was displaced towards creatinine. Above about pH 6.0 the equilibrium was associated with approximately equimolar quantities of creatine and creatinine. The creatine content of urine ranged from virtually nil to about double that of creatinine and changed predictably during storage. These findings have implications for the use of creatinine as an index of muscle mass and nutritional status, and as a marker for the completeness of urine collections.
Correspondence and requests for reprints Tennis Court Road, Cambridge CB2 lQL,
0009-8981/88/$03.50
to: N.J. Fuller, UK.
0 1988 Elsevier Science Publishers
MRC
Dunn
B.V. (Biomedical
Clinical
Nutrition
Division)
Centre,
100
200
Introduction The rate of creatinine excretion has been used to assess muscle mass in human infants, children, and adults [l]. It has also been used to provide an index of nutritional status [2] and an index of the completeness of urine collections (see Bingham and Cummings [3]). The use of creatinine for these purposes is based on the assumption that the daily rate of production of creatinine is constant, due to the presence of a constant precursor pool of creatine and creatine-phosphate located almost entirely within skeletal muscle. However, the day-to-day variation in the creatinine content of urine may be substantial. Particularly large increases in urinary creatinine levels have been reported after acute trauma [4] and inflammatory conditions [5]; and after infection [6], including experimentally induced sandfly fever in man [7]. It is surprising, despite extensive studies of creatinine metabolism over the last century, that the major increases in urine creatinine, reported in the above conditions, have not been fully explained. Physical factors such as pH and temperature are probably involved since conversion of creatine and creatine-phosphate to creatinine is thought to be via a non-enzymic process. Theoretically, this conversion may occur both within the body and in the urine. Conversions occurring in the urine may be important when the rate of creatine excretion is high as in infants [8] and in patients with muscle wasting diseases [9]. These conversions may also be important in patients suffering from acute trauma [4], and in subjects with pyrexial illnesses in whom there is excessive creatine excretion. Studies on the effect of pH and temperature on the conversion of creatine and creatine-phosphate to creatinine appear to be incomplete for the following reasons: (a) few studies have attempted to simulate the variety of conditions under which urine samples may be stored, and, on the rare occasions when they have, different buffer systems have been used with little or no overlap in pH [lo]. As a consequence there is a surprising paucity of information regarding the effect of storage conditions on creatine: creatinine interconversions; (b) the conversion of creatine to creatinine is associated with the utilization of hydrogen ions which may alter the pH of the selected buffer system, especially if it is weak; (c) the measurement of creatinine by the JaffC method which is strongly dependent on pH, may be affected by the presence of strong buffers in the test samples [ll]. In the case of urine this is usually overcome by adopting high dilution factors. It is possible that the above factors may influence the inter-conversion of creatine and creatinine to an extent hitherto unrealised; and this may have important implications for the interpretation of data involving creatinine. Therefore, we have developed assay conditions which take into account the above factors, and we have assessed the inter-conversion of creatine/creatinine over a wide range of pH and temperature, to cover both physiological conditions and the conditions to which urine samples may be subjected during storage. The main aims of the study were to (a) identify adverse storage conditions and (b) to both measure and predict the rate and extent of inter-conversions in urine samples stored under a variety of conditions.
201
Methods Materials Creatinine, creatine, and creatine-phosphate were obtained from Sigma Chemical Company Ltd., Poole, Dorset, UK. Creatinine was found to be essentially free of creatine (enzymatic method); creatine-phosphate was found to contain no measurable creatine (enzymatic method) or creatinine (Jaffe method); and creatine was found to contain about 0.3% creatinine (Jafft method). The analytical procedures are as described below. Measurement
of creatinine,
creatine, and creatine-phosphate
The creatinine concentration of samples was measured by the Jaffe method (alkaline-picrate reaction) using Kit No. 124 192, BCL, Boehringer Lewes, East Sussex, UK; and enzymatically by the method of Wahlefeld et al [12]. The creatine concentration was measured enzymatically by the method of Bernt et al [13]; it was also measured, after prior conversion to creatinine in 6N HCl, by the JaffC method
1141. Effect of pH and temperature
on the rate of inter-conversion
of creatine and creatinine
Buffered systems A buffer system (0.05 mol/l citric acid/O.05 mol/l maleic acid-adjusted with NaOH) was used to ensure adequate buffering capacity over a pH range of 2.0-7.4. Diethanolamine-HCl or Tris (hydroxymethyl) aminomethane buffer systems were used to cover pH 7.1-10.0 (from Dawson et al [15]). Creatine, creatine-phosphate, or creatinine were added to these buffers in concentrations ranging from lo-100 mmol/l. After incubation at constant temperatures (0-40°C), for periods ranging from O-24 h, the pH was determined to ensure that no change in pH had occurred as a result of creatine/creatinine inter-conversions. The mixture was then neutralized to pH 7 with 5 mol/l NaOH. Creatinine and creatine concentrations were measured, as indicated above, and their rates of inter-conversion were then determined. To ensure that the choice of these buffering systems had no quantitative effect on the rates of interconversion of creatine and creatinine, other buffers were selected for comparison, and the above procedures repeated. These included: HCl-KC1 at pH 1.0-2.2; glycine-HCl at pH 2.2-3.6; phthalate-HCl at pH 2.2-4.0; sodium acetate-acetic acid at pH 3.7-5.6; sodium cacodylate-HCl at pH 5.0-7.4; Na,HPO,-NaH,PO, at pH 5.8-7.4; triethanolamine hydrochloride-NaOH at pH 6.8-7.4; Tris (hydroxymethyl) aminomethane-HCl at pH 7.1-8.9 (all from Dawson et al [15]). In addition, the effect of buffer molarity on the rates of interconversion of creatine and creatinine was assessed, using the citrate/maleate buffer system (between O-O.5 mol/l). In all these systems the buffers were neutralized and then
202
diluted 50-fold to ensure that the JaffC reaction proceed at its intended pH [II]. Freshly prepared creatinine and creatine standards were treated in a similar way. The effect of pH (between pH 2 and 10) on the position of equilibrium between creatine and creatinine was established at certain temperatures (22” C, 37 o C, and 40’ C), to take into account the various physiological states and storage conditions to which urine may be subjected before analysis. The position of equilibrium was assessed by incubating, for prolonged periods of time, solutions containing either creatine (no creatinine initially) or creatinine (no creatine initially). The concentration of creatine and creatinine was assessed at regular intervals, until a constant ratio was shown to exist between them. Starting mixtures corresponding to the equilibrium ratios at the particular pH values (pH 2-10) which had been thus established, were incubated under identical conditions to confirm that the equilibrium positions were indeed correct, and also to ensure that the creatine and creatinine had not degraded to other substances during the prolonged incubation period. The direction and extent of the inter-conversion of creatine and creatinine, under given conditions of pH and temperature, was predicted by the following method. The formation of creatinine, in a solution containing only creatine to begin with, was calculated by multiplying the fractional rate of conversion of creatine to creatinine (fr-creatine) by the molar concentration of creatine ([creatine]). The formation of creatine, in a solution containing only creatinine to begin with was calculated by multiplying the fractional rate of conversion of creatinine to creatine (fr-creatinine) by the molar concentration of creatinine ([creatinine]). The net conversion of creatine to creatinine, in a solution containing a mixture of creatine and creatinine was calculated by the use of the following equation: Net conversion = ([ creatine]
(creatine X
to creatinine)
fr-creatine)
- ([ creatinine]
X
fr-creatinine) .
When the sign is positive, there is a predicted net gain of creatinine (and net loss of creatine), and when the sign is negative, there is a predicted net loss of creatinine (and net gain of creatine). To confirm the validity of the above equation, buffered solutions (and urine samples, see below) containing varying concentrations and ratios of creatine/ creatinine were incubated for 24 h so that the observed changes could be compared with the predicted changes.
Urine samples Random urine samples were obtained (and immediately frozen) from the following individuals: 20 healthy adults; eight premature babies, 2-wk old (born at 28-30 weeks gestation period); 10 non-pyrexial adult patients with Crohn’s disease, receiving total parenteral nutrition; six individuals undergoing partial (300 kcal/day) or total starvation for obesity; and eight adults with mild pyrexial illnesses.
203
Eighteen of these urine samples (a few of which were spiked with creatine to raise the creatine content of urine close to that of creatinine) were stored for 24 h, under various conditions of pH (pH 2.7) and temperature (20-40 o C), to establish whether or not the rate of formation, or loss, of creatinine in urine could be predicted by the same equation as used for the buffered systems. All these urine samples had thiomersal added to them (0.25 mg/20 ml) and their creatine/creatinine content measured by the nonenzymatic assay, both before and after incubation. Results
The intra-assay coefficient of variation for repeated measurements of creatinine by the Jaff6 method in both urine and standard buffei solutions was found to be 0.8% (n = 10). The inter-assay coefficient of variation was 2.9% (n = 23). The coefficient of variation for creatine as measured by the enzymatic assay was 3.2%: and by the alkaline-picrate method was 1.3%. The fractional conversion of creatine to creatinine, and vice versa, was found to be independent of concentration over the range 10 to 100 mmol/l. Therefore, the
1
2
3
4
5
6
7
PH Fig. 1. The effect of pH on the conversion rate of creatine to creatinine in 0.05 mol/l mol/l sodium maleate buffer. The initial solutions contained no creatinine.
sodium
citrate/O.05
,-
I-
i-
3 --
0
5
10
15
20
25
Temperature
30
Fig. 2. The effect of temperature on the conversion to creatinine in a 0.05 mol/l sodium citrate/O.05
contained
35
40
“C
rate of creatine to creatinine and creatine-phosphate mol/l sodium maleate buffer. The initial solutions
no creatinine.
0
I
I
2
4
I 6
I
6
1 10
PH
Fig. 3. The effect of pH on the conversion (see text for details). The initial solutions
rate of creatinine to creatine contained no creatine.
at 37 o C in various
buffer
systems
205
100-
a0 Q
E
60-
Z :
3
40-
E 20-
0! 0
1
2
3
4
5
6
7
a
9
10
PH
Fig. 4. The effect of pH on the position of equilibrium for a mixture of creatine and creatinine at 37 o C in various buffer systems (see text for details). The percentage of creatinine in the eq~ilib~um mixture is shown. The difference between 100% and the creatinine percentage gives the creatine percentage at equilibrium.
extent of inter-conversion between creatine and creatinine is given in percentage terms because of its more universal application. The effects of pH, at selected temperatures, on the percentage conversion of creatine to creatinine, for a 24 h period, is shown in Fig. 1. The maximum rate of conversion of creatine to creatinine was found to occur between pH 3.5 and 4.0. The reaction is first order with respect to creatine, and temperature dependent (Fig. 2); at temperatures below 4’ C the rate of conversion is negligible, and undetectable in solutions that had been frozen. The percentage conversion of creatinine to creatine over a range of pHs is shown in Fig. 3. The m~mum rate of reaction occurs at about pH 5. Between pH 6 and pH 10 the rate of conversion of creatinine to creatine (Fig. 3) is approximately the same as the rate of conversion of creatine to creatinine (data not shown). These findings suggest that equimolar ratios of creatine and creatinine should exist at equilibrium. This was confirmed experimentally (Fig. 4). The effect of pH at various temperatures on the fractional rate of conversion of creatine-phosphate to creatinine was found to be similar to, but slightly faster than, the rate of conversion of creatine to creatinine (data not shown). Neither the concentration or the type of buffer used were found to have an effect on the rate of interconversion of creatine and creatinine, unless the change in creatimne (which is more basic than creatine) was sufficient to alter pH of the buffer (this only occurred in weak buffers - weaker than those normally found in urine). However, it should be noted that the rate of the Jaffe reaction was found to be affected by the presence of strong buffers, and therefore, all assays were carried out after diluting the buffer as described in the ‘methods’ section. Urine samples spiked with creatine (O-13.8 mmol/l) showed no change in pH when incubated for 24 h. under various conditions.
206
The net rate of conversion of creatine to creatinine, and vice versa, gradually decreased as the mixture approached equilibrium. The same ultimate equilibrium position was obtained irrespective of whether creatine or creatinine alone, or a mixture of the two, were used in the original solutions (Fig. 4). Temperature (22-40 o C) had little, if any, effect on the equilibrium position.
Urine samples
The recovery
of exogenous creatinine added to urine was found to be 99.0% of added creatine determined by the alkaline-picrate method was found to be 103 h 2.3% (n = 9) and by the enzymatic assay was found to be 95.0 + 5.3% (n = 7). The variation in the molar ratio of creatine/creatinine in samples of urine obtained from individuals with a variety of conditions are indicated in Table I. Values obtained from a number of other studies are also included for comparison. In this study only two out of the twenty normal urine samples contained creatine and creatinine in a molar ratio above 0.10. None of the urine samples obtained from patients receiving total parental nutrition had molar ratios above 0.04. Three out of eight of the individuals with pyrexial illness produced urine that had a molar ratio in excess of 0.10 (overall range 0.001-0.370). Of the eight premature baby samples one had a ratio of 1.33, two of 0.6 and three between 0.2 and 0.6. In obese individuals receiving a very low calorie diet for 1 wk, the molar ratio of creatine: creatinine rose from about 0.1 (range o-0.25) to 0.4 (range 0.22-0.65); but there was no further change after an additional six days of total starvation. (n = 10). The recovery
TABLE
I
The range conditions
of urinary
creatine:
creatinine
molar
ratios
for normal
subjects
or in some
Condition
Creatine/Creatinine
Norma1 adult males
]9, 16,171 (Present study)
Children Premature infants Trauma + elective surgery Pyrexia Starvation Total parenteral nutrition Thyrotoxicosis Muscular disorders Methyl testosterone
0 -0.07 0.001-0.075 o-o.41 0.005-0.19 0.09-2.0 0.04-1.33 0.31-1.38 0.001-0.37 0.61-0.77 0.003-0.044 0.10-1.25 0.29-2.00 2.0
Normal mice [67-135 days of age] Dystrophic mice (67-135 days of age)
1.15 2.26
]211 ]211
Normal
adult females
molar ratio (range)
]L6, 171 (Present study) ]8, 161 (Present
study)
]41 (Present (Present (Present
study) study, [18]) study)
]17,191 191 ]201
pathological
207 25
1
I
-5
0
0
A
. Buffered
5
10
Predicted
Fig. 5. A comparison between the buffered solutions (O), and urine various pathophysiological ratios (see text for details). A positive negative sign indicates a decrease
15
20
samples
25
% change
predicted and observed percentage changes in the creatinine content of samples (0) during 24 h of storage. The samples, which contained of creatine to creatinine, were kept at various pHs and temperatures sign indicates an increase in creatinine (decrease in creatine) and a in creatinine (increase in creatine).
Predicted verse observed changes The quantitative changes observed in urine samples was the same as would be predicted from the buffered test systems, provided that the starting mixture is known. As with the buffered system the proportional changes were independent of concentration. Therefore, the results obtained from urine samples have also been expressed in terms of percentage inter-conversions. Figure 5 shows that there is good agreement between the observed and predicted changes in creatinine concentration in both urine and artificially buffered samples stored for 24 h under various conditions. Urine samples spiked with creatine also behaved in a predictable manner. Discussion The study has quantified the effect of pH and temperature on the position of equilibrium and on the rate of inter-conversion of creatine and creatinine. The results obtained in vitro adequately predict the changes which occur in urine samples under various conditions. The study also suggests that major errors might arise in the determination of both urinary creatine and creatinine as a result of adverse storage conditions.
208
The creatine-creatinine equilibrium position The creatine-creatinine equilibrium position was found to be essentially independent of temperature between 22 o and 40 o C, but to be strongly dependent on pH (Fig. 4). In comparison with the data obtained by Edgar and Shiver (10) at 50°C this study shows the position of equilibrium to be displaced by about one pH unit to the right. Our results suggest that at low pH (pH < 2) virtually all of the creatine in a creatine-creatinine mixture will be ultimately converted to creatinine. At pH > 6, approximately equimolar ratios of creatine and creatinine will ultimately be established. Thus a sterile urine sample containing only creatinine initially will lose half of its creatinine content on prolonged storage at pH > 6. However, it must be emphasized that the position of the equilibrium gives no indication of the absolute rate at which mixtures of creatine and creatinine proceed towards their equilibrium position. The rate of creatine-creatinine inter-conversion The net rate of inter-conversion of creatine and creatinine is shown to depend on temperature, pH, and initial ratio of creatine and creatinine. Our results indicate that the maximum rate of conversion of creatine to creatinine occurs between pH 3.5 and 4.0 (in contrast to the belief of Edgar and Wakefield [22], that the lower the pH, the faster the reaction will proceed from creatine to creatinine). The maximum rate of conversion of creatinine to creatine was found to occur at about pH 5. Reference to Figs. l-3 allows a prediction to be made of the rate of gain or loss of creatinine and creatine, from solutions containing either or both substances. The predicted changes for both buffered solutions and urine samples, stored under various conditions, agree closely with experimentally obtained changes (Fig. 5). Our results also allow a prediction to be made of the molar ratios of creatine and creatinine which will allow the rate of the forward reaction to equal the rate of the reverse reaction. These predictions again agree closely with the experimentally obtained ratios of creatine and creatinine at equilibrium. The rate of creatine-creatinine inter-conversions was also found, as expected, to be strongly dependent on temperature. At low temperatures, the rate of interconversion was considerably slower than at higher temperatures. In frozen samples, of both urine and buffers, no significant changes were observed. These particular findings are at variance with those of Soliman et al [23], who claim that there are measurable reductions in creatinine concentrations in acidified urine samples stored in a frozen state for periods as short as 24 h. Clinical relevance of results Since temperature affects the rate of interconversion of creatine and creatinine at all pHs measured, the present work is of relevance, not only to the storage of urine samples, but also to the production of creatinine from both creatine and creatinephosphate in vivo. A 3” C rise in body temperature, which may occur during infection, may increase the daily production of creatinine (from a constant creatine/creatinine-phosphate pool) by 15-20s. Therefore, it is not surprising that a rise in creatinine excretion has been reported during pyrexial illness [5,6,7]. However, it should be noted that in these [5,6,7] and other studies [1,2,4] it is assumed
209
that the creatinine measured in urine represents the creatinine which is actually excreted. It is unfortunate that in many of these studies there is a paucity of information regarding the creatine content of the urine samples, and the storage conditions (including pH, temperature, preservatives - if any, and length of time of storage) because they may help to explain the very large apparent increases in creatinine excretion, which have occasionally been reported to double. For example, when a urine sample containing equimolar concentrations of creatine and creatinine (which may be obtained from normal children, or subjects suffering from acute injury or muscle disease) is stored at pH 3.5-4.0 and a temperature of 37 o C, for 24 h, its creatinine content will rise by about 20%. Longer periods of storage will be associated with a greater rise in creatinine. It is also possible for the creatinine content of urine to decrease during storage. For instance, the creatinine content of a urine sample from an adult male containing little or no creatine initially, will show a decrease of about 5% when stored at pH 5 and 37’C, for 24 h (see Fig. 3). Tanzer and Gilvarg [24] also suggested that the creatinine content of urine may decrease during storage, whilst its creatine content increases. Furthermore, they suggested that this can occur whilst urine is in the bladder. However, quantitative calculations based on the present results suggest that the maximum decrease in creatinine during bladder storage is likely to be small (2-3% over 12 h). On the other hand, as much as 50% of creatinine will be ultimately lost from a sterile urine sample stored at pH > 6 (Fig. 4). These changes are of significance to urine samples stored for prolonged periods of time at high temperatures, which may occur, for example, in laboratories and field stations in the Third World. Clearly, the creatine and creatinine content of urine may either rise or fall during storage, depending on the initial molar ratio of these two interconvertible substances, and on the storage conditions, which may vary greatly. The pH of stored urine may range from pH O-2, when strong acids are used as preservatives (eg 10 ml of concentrated HCl for a 24 h urine collection of an adult), pH 2-5 when boric acid or small amounts of HCl are used as preservatives (eg 50 ml of 15% HCl [25]), pH 5-8 when freshly voided urine is collected (with or without bacteriostatic agents such as thiomersal), and pH > 8 when alkali is added to urine. The length and temperature of storage at these pHs, which may vary considerably, and also influence the interpretation of urinary creatine/creatinine results. In summary, the present study confirms the wide variation in the ranges of initial molar ratios of creatine/creatinine in urine (Table I). In addition, it allows a satisfactory prediction to be made of the changes which may occur as a result of storage under a variety of conditions of pH and temperature. These findings, together with the increased production of creatinine which is likely to occur in subjects with pyrexial illnesses, are of importance to nutritional, renal, and metabolic balance studies in both humans and animals, where creatinine is used for the assessment of muscle mass, renal function and the completeness of urine collections. Acknowledgement The authors
thank
Dr. 1. Williams
for helpful
advice during
this study.
210
References 1 Forbes GB, Bruining GJ. Urinary creatinine excretion and lean body mass. Am J Clin Nutr 1976;29:1359-1366. 2 Bistrian BR, Blackburn GL, Sherman M, Scrimshaw NS. Therapeutic index of nutritional depletion in hospitalized patients. Surg Gynecol Obstet 1975;141:512-516. 3 Bingham SA, Cummings JH. The use of creatinine output as a check on the completeness of 24 hour urine collections. Human Nutr: Clin Nutr 1985;39(7:343-53. 4 Threlfall CJ, Stoner HB, Galasko CSB. Patterns in the excretion of muscle markers after trauma and orthopedic surgery. J Trauma 1981;21:140-147. 5 Schiller WR, Long CL, Blakemore WS. Creatinine and nitrogen excretion in seriously ill and injured patients. Surg Gynecol Obstet 1979;149:561-566. 6 Ton&ins AM, Garlick PJ, Schofield WN, Waterlow JC. ‘The combined effects of infection and malnutrition on protein metabolism in children. Clin Sci 1983;65:313-324. I Wannemacher RW, Dinterman RE, Pekarek RS, Bartelloni PJ, Beisel WR. Urinary ammo acid excretion during experimentally induced sandfly fever in man. Am J Clin Nutr 1975;28:110-118. 8 Clark LC, Thompson HL, Beck EI, Jacobson W. Excretion of creatine and creatinine by children. Am J Dis Child 1951;81:774-783. compounds in blood and urine of patients suffering from 9 Van Pilsum JF, Wolin EA. Guanidium muscle disorders. J Lab Clin Med 1958;51:219-223. 10 Edgar G, Shiver HE. The equilibrium between creatine and creatinine in aqueous solution. The effect of hydrogen ion. J Am Chem Sot 1925;47:1179-1188. 11 Spencer K. Analytical reviews in clinical biochemistry: the estimation of creatinine. Ann Clin Biochem 1986;23:1-25. In: Bergmeyer HU, ed. Methods of enzymatic 12 Wahlefeld AW, Holz G, Bergmeyer HU. Creatinine. analysis, Vol 4. New York, San Francisco, London: Academic Press, 1974;1786-1790. In: Bergmeyer HU, ed. Methods of enzymatic 13 Bernt E, Bergmeyer HU, Mollering H. Creatine. analysis, Vol 4. New York, San Francisco, London: Academic Press, 1974;1772-1776. determination and distribution of urinary creatinine and creatine. Clin Chem 14 Clarke JT. Calorimetric 1961;7:371-383. research, 2nd ed. 15 Dawson RMC, Elliot DC, Elliot WH, Jones KM, (eds). Data for biochemical Oxford: Clarendon Press, 1968;475-508. tables Vol 1. Urine-nitrogenous substances, 8th ed. Basle: Ciba-Geigy Ltd., 16 Geigy scientific 1981;62-78. 17 Tiemey NA, Peters JP. The mode of excretion of creatine and creatine metabolism in thyroid disease. J Clin Invest 1943;22:595-602. 18 Keys A, Brozek J, Henschel A, Mickelsen 0, Taylor HL, eds. The biology of human starvation, Vol I. Minneapolis: Minnesota University Press 1950;432-438. W. Effects of thyroid on creatine metabolism with a discussion of the 19 Wilkins L, Fleischmamr mechanism of storage and excretion of creatine bodies. J Clin Invest 1946;25:360-377. 20 Wilkins L, Fleischmann W. Studies on the creatinuria due to methylated steroids. J Clin Invest 1945;24:21-32. 21 Fitch CD, Oates JD, Dinning JS. The metabolism of creatine-l-Cl4 by mice with hereditary muscular dystrophy. J Clin Invest 1961;40:850-856. 22 Edgar G, Wakefield RA. The kinetics of the conversion of creatine into creatinine in hydrochloric acid solutions. J Am Chem Sot 1923;45:2242-2245. 23 Soliman SA, Abdel-Hay MH, Sulaiman MI, Tayeb OS. Stability of creatinine, urea and uric acid in urine stored under various conditions. Clin Chim Acta 1986;160:319-326. 24 Tamer ML, Gilvarg C. Creatine and creatine kinase measurement. J Biol Chem 1959;234:3201-3204. 25 Albanese AA, Wangerin DM. The creatine and creatinine excretion of normal adult males. Science 1944;100:58-60.
199
CCA 04223
Factors influencing the production of creatinine: implications for the determination and interpretation of urinary creatinine and creatine in man Nigel J Fuller and Marinos MRC Dunn Clinical Nutrition (Received
Elia
Centre, Cambridge (UK)
20 October 1987; revision received 26 February accepted after revision 29 February 1988)
Key wordst Creatinine;
Creatine;
Creatine-phosphate;
Urine;
1988;
Equilibrium
Summary
The rates of creatine/creatinine inter-conversions and their equilibrium were studied under controlled conditions of temperature and pH that simulate urine storage conditions. The concentrations and ratios of creatine to creatinine in urine obtained from subjects with various pathophysiological conditions were determined, both before and after storage. The observed changes occurring during storage were compared with predicted changes based on observations of standard solutions. The initial reaction rate was found to increase with temperature, occurring maximally at about pH 3.7 for the conversion of creatine to creatinine, and at about pH 5.0 for the conversion of creatinine to creatine. At low pHs the equilibrium position was displaced towards creatinine. Above about pH 6.0 the equilibrium was associated with approximately equimolar quantities of creatine and creatinine. The creatine content of urine ranged from virtually nil to about double that of creatinine and changed predictably during storage. These findings have implications for the use of creatinine as an index of muscle mass and nutritional status, and as a marker for the completeness of urine collections.
Correspondence and requests for reprints Tennis Court Road, Cambridge CB2 lQL,
0009-8981/88/$03.50
to: N.J. Fuller, UK.
0 1988 Elsevier Science Publishers
MRC
Dunn
B.V. (Biomedical
Clinical
Nutrition
Division)
Centre,
100
200
Introduction The rate of creatinine excretion has been used to assess muscle mass in human infants, children, and adults [l]. It has also been used to provide an index of nutritional status [2] and an index of the completeness of urine collections (see Bingham and Cummings [3]). The use of creatinine for these purposes is based on the assumption that the daily rate of production of creatinine is constant, due to the presence of a constant precursor pool of creatine and creatine-phosphate located almost entirely within skeletal muscle. However, the day-to-day variation in the creatinine content of urine may be substantial. Particularly large increases in urinary creatinine levels have been reported after acute trauma [4] and inflammatory conditions [5]; and after infection [6], including experimentally induced sandfly fever in man [7]. It is surprising, despite extensive studies of creatinine metabolism over the last century, that the major increases in urine creatinine, reported in the above conditions, have not been fully explained. Physical factors such as pH and temperature are probably involved since conversion of creatine and creatine-phosphate to creatinine is thought to be via a non-enzymic process. Theoretically, this conversion may occur both within the body and in the urine. Conversions occurring in the urine may be important when the rate of creatine excretion is high as in infants [8] and in patients with muscle wasting diseases [9]. These conversions may also be important in patients suffering from acute trauma [4], and in subjects with pyrexial illnesses in whom there is excessive creatine excretion. Studies on the effect of pH and temperature on the conversion of creatine and creatine-phosphate to creatinine appear to be incomplete for the following reasons: (a) few studies have attempted to simulate the variety of conditions under which urine samples may be stored, and, on the rare occasions when they have, different buffer systems have been used with little or no overlap in pH [lo]. As a consequence there is a surprising paucity of information regarding the effect of storage conditions on creatine: creatinine interconversions; (b) the conversion of creatine to creatinine is associated with the utilization of hydrogen ions which may alter the pH of the selected buffer system, especially if it is weak; (c) the measurement of creatinine by the JaffC method which is strongly dependent on pH, may be affected by the presence of strong buffers in the test samples [ll]. In the case of urine this is usually overcome by adopting high dilution factors. It is possible that the above factors may influence the inter-conversion of creatine and creatinine to an extent hitherto unrealised; and this may have important implications for the interpretation of data involving creatinine. Therefore, we have developed assay conditions which take into account the above factors, and we have assessed the inter-conversion of creatine/creatinine over a wide range of pH and temperature, to cover both physiological conditions and the conditions to which urine samples may be subjected during storage. The main aims of the study were to (a) identify adverse storage conditions and (b) to both measure and predict the rate and extent of inter-conversions in urine samples stored under a variety of conditions.
201
Methods Materials Creatinine, creatine, and creatine-phosphate were obtained from Sigma Chemical Company Ltd., Poole, Dorset, UK. Creatinine was found to be essentially free of creatine (enzymatic method); creatine-phosphate was found to contain no measurable creatine (enzymatic method) or creatinine (Jaffe method); and creatine was found to contain about 0.3% creatinine (Jafft method). The analytical procedures are as described below. Measurement
of creatinine,
creatine, and creatine-phosphate
The creatinine concentration of samples was measured by the Jaffe method (alkaline-picrate reaction) using Kit No. 124 192, BCL, Boehringer Lewes, East Sussex, UK; and enzymatically by the method of Wahlefeld et al [12]. The creatine concentration was measured enzymatically by the method of Bernt et al [13]; it was also measured, after prior conversion to creatinine in 6N HCl, by the JaffC method
1141. Effect of pH and temperature
on the rate of inter-conversion
of creatine and creatinine
Buffered systems A buffer system (0.05 mol/l citric acid/O.05 mol/l maleic acid-adjusted with NaOH) was used to ensure adequate buffering capacity over a pH range of 2.0-7.4. Diethanolamine-HCl or Tris (hydroxymethyl) aminomethane buffer systems were used to cover pH 7.1-10.0 (from Dawson et al [15]). Creatine, creatine-phosphate, or creatinine were added to these buffers in concentrations ranging from lo-100 mmol/l. After incubation at constant temperatures (0-40°C), for periods ranging from O-24 h, the pH was determined to ensure that no change in pH had occurred as a result of creatine/creatinine inter-conversions. The mixture was then neutralized to pH 7 with 5 mol/l NaOH. Creatinine and creatine concentrations were measured, as indicated above, and their rates of inter-conversion were then determined. To ensure that the choice of these buffering systems had no quantitative effect on the rates of interconversion of creatine and creatinine, other buffers were selected for comparison, and the above procedures repeated. These included: HCl-KC1 at pH 1.0-2.2; glycine-HCl at pH 2.2-3.6; phthalate-HCl at pH 2.2-4.0; sodium acetate-acetic acid at pH 3.7-5.6; sodium cacodylate-HCl at pH 5.0-7.4; Na,HPO,-NaH,PO, at pH 5.8-7.4; triethanolamine hydrochloride-NaOH at pH 6.8-7.4; Tris (hydroxymethyl) aminomethane-HCl at pH 7.1-8.9 (all from Dawson et al [15]). In addition, the effect of buffer molarity on the rates of interconversion of creatine and creatinine was assessed, using the citrate/maleate buffer system (between O-O.5 mol/l). In all these systems the buffers were neutralized and then
202
diluted 50-fold to ensure that the JaffC reaction proceed at its intended pH [II]. Freshly prepared creatinine and creatine standards were treated in a similar way. The effect of pH (between pH 2 and 10) on the position of equilibrium between creatine and creatinine was established at certain temperatures (22” C, 37 o C, and 40’ C), to take into account the various physiological states and storage conditions to which urine may be subjected before analysis. The position of equilibrium was assessed by incubating, for prolonged periods of time, solutions containing either creatine (no creatinine initially) or creatinine (no creatine initially). The concentration of creatine and creatinine was assessed at regular intervals, until a constant ratio was shown to exist between them. Starting mixtures corresponding to the equilibrium ratios at the particular pH values (pH 2-10) which had been thus established, were incubated under identical conditions to confirm that the equilibrium positions were indeed correct, and also to ensure that the creatine and creatinine had not degraded to other substances during the prolonged incubation period. The direction and extent of the inter-conversion of creatine and creatinine, under given conditions of pH and temperature, was predicted by the following method. The formation of creatinine, in a solution containing only creatine to begin with, was calculated by multiplying the fractional rate of conversion of creatine to creatinine (fr-creatine) by the molar concentration of creatine ([creatine]). The formation of creatine, in a solution containing only creatinine to begin with was calculated by multiplying the fractional rate of conversion of creatinine to creatine (fr-creatinine) by the molar concentration of creatinine ([creatinine]). The net conversion of creatine to creatinine, in a solution containing a mixture of creatine and creatinine was calculated by the use of the following equation: Net conversion = ([ creatine]
(creatine X
to creatinine)
fr-creatine)
- ([ creatinine]
X
fr-creatinine) .
When the sign is positive, there is a predicted net gain of creatinine (and net loss of creatine), and when the sign is negative, there is a predicted net loss of creatinine (and net gain of creatine). To confirm the validity of the above equation, buffered solutions (and urine samples, see below) containing varying concentrations and ratios of creatine/ creatinine were incubated for 24 h so that the observed changes could be compared with the predicted changes.
Urine samples Random urine samples were obtained (and immediately frozen) from the following individuals: 20 healthy adults; eight premature babies, 2-wk old (born at 28-30 weeks gestation period); 10 non-pyrexial adult patients with Crohn’s disease, receiving total parenteral nutrition; six individuals undergoing partial (300 kcal/day) or total starvation for obesity; and eight adults with mild pyrexial illnesses.
203
Eighteen of these urine samples (a few of which were spiked with creatine to raise the creatine content of urine close to that of creatinine) were stored for 24 h, under various conditions of pH (pH 2.7) and temperature (20-40 o C), to establish whether or not the rate of formation, or loss, of creatinine in urine could be predicted by the same equation as used for the buffered systems. All these urine samples had thiomersal added to them (0.25 mg/20 ml) and their creatine/creatinine content measured by the nonenzymatic assay, both before and after incubation. Results
The intra-assay coefficient of variation for repeated measurements of creatinine by the Jaff6 method in both urine and standard buffei solutions was found to be 0.8% (n = 10). The inter-assay coefficient of variation was 2.9% (n = 23). The coefficient of variation for creatine as measured by the enzymatic assay was 3.2%: and by the alkaline-picrate method was 1.3%. The fractional conversion of creatine to creatinine, and vice versa, was found to be independent of concentration over the range 10 to 100 mmol/l. Therefore, the
1
2
3
4
5
6
7
PH Fig. 1. The effect of pH on the conversion rate of creatine to creatinine in 0.05 mol/l mol/l sodium maleate buffer. The initial solutions contained no creatinine.
sodium
citrate/O.05
,-
I-
i-
3 --
0
5
10
15
20
25
Temperature
30
Fig. 2. The effect of temperature on the conversion to creatinine in a 0.05 mol/l sodium citrate/O.05
contained
35
40
“C
rate of creatine to creatinine and creatine-phosphate mol/l sodium maleate buffer. The initial solutions
no creatinine.
0
I
I
2
4
I 6
I
6
1 10
PH
Fig. 3. The effect of pH on the conversion (see text for details). The initial solutions
rate of creatinine to creatine contained no creatine.
at 37 o C in various
buffer
systems
205
100-
a0 Q
E
60-
Z :
3
40-
E 20-
0! 0
1
2
3
4
5
6
7
a
9
10
PH
Fig. 4. The effect of pH on the position of equilibrium for a mixture of creatine and creatinine at 37 o C in various buffer systems (see text for details). The percentage of creatinine in the eq~ilib~um mixture is shown. The difference between 100% and the creatinine percentage gives the creatine percentage at equilibrium.
extent of inter-conversion between creatine and creatinine is given in percentage terms because of its more universal application. The effects of pH, at selected temperatures, on the percentage conversion of creatine to creatinine, for a 24 h period, is shown in Fig. 1. The maximum rate of conversion of creatine to creatinine was found to occur between pH 3.5 and 4.0. The reaction is first order with respect to creatine, and temperature dependent (Fig. 2); at temperatures below 4’ C the rate of conversion is negligible, and undetectable in solutions that had been frozen. The percentage conversion of creatinine to creatine over a range of pHs is shown in Fig. 3. The m~mum rate of reaction occurs at about pH 5. Between pH 6 and pH 10 the rate of conversion of creatinine to creatine (Fig. 3) is approximately the same as the rate of conversion of creatine to creatinine (data not shown). These findings suggest that equimolar ratios of creatine and creatinine should exist at equilibrium. This was confirmed experimentally (Fig. 4). The effect of pH at various temperatures on the fractional rate of conversion of creatine-phosphate to creatinine was found to be similar to, but slightly faster than, the rate of conversion of creatine to creatinine (data not shown). Neither the concentration or the type of buffer used were found to have an effect on the rate of interconversion of creatine and creatinine, unless the change in creatimne (which is more basic than creatine) was sufficient to alter pH of the buffer (this only occurred in weak buffers - weaker than those normally found in urine). However, it should be noted that the rate of the Jaffe reaction was found to be affected by the presence of strong buffers, and therefore, all assays were carried out after diluting the buffer as described in the ‘methods’ section. Urine samples spiked with creatine (O-13.8 mmol/l) showed no change in pH when incubated for 24 h. under various conditions.
206
The net rate of conversion of creatine to creatinine, and vice versa, gradually decreased as the mixture approached equilibrium. The same ultimate equilibrium position was obtained irrespective of whether creatine or creatinine alone, or a mixture of the two, were used in the original solutions (Fig. 4). Temperature (22-40 o C) had little, if any, effect on the equilibrium position.
Urine samples
The recovery
of exogenous creatinine added to urine was found to be 99.0% of added creatine determined by the alkaline-picrate method was found to be 103 h 2.3% (n = 9) and by the enzymatic assay was found to be 95.0 + 5.3% (n = 7). The variation in the molar ratio of creatine/creatinine in samples of urine obtained from individuals with a variety of conditions are indicated in Table I. Values obtained from a number of other studies are also included for comparison. In this study only two out of the twenty normal urine samples contained creatine and creatinine in a molar ratio above 0.10. None of the urine samples obtained from patients receiving total parental nutrition had molar ratios above 0.04. Three out of eight of the individuals with pyrexial illness produced urine that had a molar ratio in excess of 0.10 (overall range 0.001-0.370). Of the eight premature baby samples one had a ratio of 1.33, two of 0.6 and three between 0.2 and 0.6. In obese individuals receiving a very low calorie diet for 1 wk, the molar ratio of creatine: creatinine rose from about 0.1 (range o-0.25) to 0.4 (range 0.22-0.65); but there was no further change after an additional six days of total starvation. (n = 10). The recovery
TABLE
I
The range conditions
of urinary
creatine:
creatinine
molar
ratios
for normal
subjects
or in some
Condition
Creatine/Creatinine
Norma1 adult males
]9, 16,171 (Present study)
Children Premature infants Trauma + elective surgery Pyrexia Starvation Total parenteral nutrition Thyrotoxicosis Muscular disorders Methyl testosterone
0 -0.07 0.001-0.075 o-o.41 0.005-0.19 0.09-2.0 0.04-1.33 0.31-1.38 0.001-0.37 0.61-0.77 0.003-0.044 0.10-1.25 0.29-2.00 2.0
Normal mice [67-135 days of age] Dystrophic mice (67-135 days of age)
1.15 2.26
]211 ]211
Normal
adult females
molar ratio (range)
]L6, 171 (Present study) ]8, 161 (Present
study)
]41 (Present (Present (Present
study) study, [18]) study)
]17,191 191 ]201
pathological
207 25
1
I
-5
0
0
A
. Buffered
5
10
Predicted
Fig. 5. A comparison between the buffered solutions (O), and urine various pathophysiological ratios (see text for details). A positive negative sign indicates a decrease
15
20
samples
25
% change
predicted and observed percentage changes in the creatinine content of samples (0) during 24 h of storage. The samples, which contained of creatine to creatinine, were kept at various pHs and temperatures sign indicates an increase in creatinine (decrease in creatine) and a in creatinine (increase in creatine).
Predicted verse observed changes The quantitative changes observed in urine samples was the same as would be predicted from the buffered test systems, provided that the starting mixture is known. As with the buffered system the proportional changes were independent of concentration. Therefore, the results obtained from urine samples have also been expressed in terms of percentage inter-conversions. Figure 5 shows that there is good agreement between the observed and predicted changes in creatinine concentration in both urine and artificially buffered samples stored for 24 h under various conditions. Urine samples spiked with creatine also behaved in a predictable manner. Discussion The study has quantified the effect of pH and temperature on the position of equilibrium and on the rate of inter-conversion of creatine and creatinine. The results obtained in vitro adequately predict the changes which occur in urine samples under various conditions. The study also suggests that major errors might arise in the determination of both urinary creatine and creatinine as a result of adverse storage conditions.
208
The creatine-creatinine equilibrium position The creatine-creatinine equilibrium position was found to be essentially independent of temperature between 22 o and 40 o C, but to be strongly dependent on pH (Fig. 4). In comparison with the data obtained by Edgar and Shiver (10) at 50°C this study shows the position of equilibrium to be displaced by about one pH unit to the right. Our results suggest that at low pH (pH < 2) virtually all of the creatine in a creatine-creatinine mixture will be ultimately converted to creatinine. At pH > 6, approximately equimolar ratios of creatine and creatinine will ultimately be established. Thus a sterile urine sample containing only creatinine initially will lose half of its creatinine content on prolonged storage at pH > 6. However, it must be emphasized that the position of the equilibrium gives no indication of the absolute rate at which mixtures of creatine and creatinine proceed towards their equilibrium position. The rate of creatine-creatinine inter-conversion The net rate of inter-conversion of creatine and creatinine is shown to depend on temperature, pH, and initial ratio of creatine and creatinine. Our results indicate that the maximum rate of conversion of creatine to creatinine occurs between pH 3.5 and 4.0 (in contrast to the belief of Edgar and Wakefield [22], that the lower the pH, the faster the reaction will proceed from creatine to creatinine). The maximum rate of conversion of creatinine to creatine was found to occur at about pH 5. Reference to Figs. l-3 allows a prediction to be made of the rate of gain or loss of creatinine and creatine, from solutions containing either or both substances. The predicted changes for both buffered solutions and urine samples, stored under various conditions, agree closely with experimentally obtained changes (Fig. 5). Our results also allow a prediction to be made of the molar ratios of creatine and creatinine which will allow the rate of the forward reaction to equal the rate of the reverse reaction. These predictions again agree closely with the experimentally obtained ratios of creatine and creatinine at equilibrium. The rate of creatine-creatinine inter-conversions was also found, as expected, to be strongly dependent on temperature. At low temperatures, the rate of interconversion was considerably slower than at higher temperatures. In frozen samples, of both urine and buffers, no significant changes were observed. These particular findings are at variance with those of Soliman et al [23], who claim that there are measurable reductions in creatinine concentrations in acidified urine samples stored in a frozen state for periods as short as 24 h. Clinical relevance of results Since temperature affects the rate of interconversion of creatine and creatinine at all pHs measured, the present work is of relevance, not only to the storage of urine samples, but also to the production of creatinine from both creatine and creatinephosphate in vivo. A 3” C rise in body temperature, which may occur during infection, may increase the daily production of creatinine (from a constant creatine/creatinine-phosphate pool) by 15-20s. Therefore, it is not surprising that a rise in creatinine excretion has been reported during pyrexial illness [5,6,7]. However, it should be noted that in these [5,6,7] and other studies [1,2,4] it is assumed
209
that the creatinine measured in urine represents the creatinine which is actually excreted. It is unfortunate that in many of these studies there is a paucity of information regarding the creatine content of the urine samples, and the storage conditions (including pH, temperature, preservatives - if any, and length of time of storage) because they may help to explain the very large apparent increases in creatinine excretion, which have occasionally been reported to double. For example, when a urine sample containing equimolar concentrations of creatine and creatinine (which may be obtained from normal children, or subjects suffering from acute injury or muscle disease) is stored at pH 3.5-4.0 and a temperature of 37 o C, for 24 h, its creatinine content will rise by about 20%. Longer periods of storage will be associated with a greater rise in creatinine. It is also possible for the creatinine content of urine to decrease during storage. For instance, the creatinine content of a urine sample from an adult male containing little or no creatine initially, will show a decrease of about 5% when stored at pH 5 and 37’C, for 24 h (see Fig. 3). Tanzer and Gilvarg [24] also suggested that the creatinine content of urine may decrease during storage, whilst its creatine content increases. Furthermore, they suggested that this can occur whilst urine is in the bladder. However, quantitative calculations based on the present results suggest that the maximum decrease in creatinine during bladder storage is likely to be small (2-3% over 12 h). On the other hand, as much as 50% of creatinine will be ultimately lost from a sterile urine sample stored at pH > 6 (Fig. 4). These changes are of significance to urine samples stored for prolonged periods of time at high temperatures, which may occur, for example, in laboratories and field stations in the Third World. Clearly, the creatine and creatinine content of urine may either rise or fall during storage, depending on the initial molar ratio of these two interconvertible substances, and on the storage conditions, which may vary greatly. The pH of stored urine may range from pH O-2, when strong acids are used as preservatives (eg 10 ml of concentrated HCl for a 24 h urine collection of an adult), pH 2-5 when boric acid or small amounts of HCl are used as preservatives (eg 50 ml of 15% HCl [25]), pH 5-8 when freshly voided urine is collected (with or without bacteriostatic agents such as thiomersal), and pH > 8 when alkali is added to urine. The length and temperature of storage at these pHs, which may vary considerably, and also influence the interpretation of urinary creatine/creatinine results. In summary, the present study confirms the wide variation in the ranges of initial molar ratios of creatine/creatinine in urine (Table I). In addition, it allows a satisfactory prediction to be made of the changes which may occur as a result of storage under a variety of conditions of pH and temperature. These findings, together with the increased production of creatinine which is likely to occur in subjects with pyrexial illnesses, are of importance to nutritional, renal, and metabolic balance studies in both humans and animals, where creatinine is used for the assessment of muscle mass, renal function and the completeness of urine collections. Acknowledgement The authors
thank
Dr. 1. Williams
for helpful
advice during
this study.
210
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