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Technical Briefs |
1 Laboratory of Biochemistry and
2
Department of Internal Medicine, University Hospital, University of Ioannina, Medical School, 455 00 Ioannina, Greece
aauthor for correspondence; fax 30-6510-99418, e-mail ebairakt{at}cc.uoi.gr
Rhabdomyolysis is a common clinical and laboratory syndrome, resulting from skeletal muscle injury, that leads to the release of potentially toxic muscle cell contents, specifically myoglobin, into the plasma (1)(2). Rhabdomyolysis can be induced by many different mechanisms and has been implicated as a major cause of acute renal failure. However, when correct treatment is provided, it is usually reversible. Acute tubular necrosis after rhabdomyolysis does not always appear to parallel the degree of muscle damage, but it may be related to other factors that potentiate the effects of myoglobinuria, such as hypotension, acidosis, and volume depletion. The pathophysiology of the renal injury is not fully understood, but it probably includes a combination of hypoperfusion/ischemia, iron tubular cytotoxicity, and cast formation potentially leading to proximal tubular necrosis and acute renal failure.
The combination of fibrates plus 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) is an effective and generally well-tolerated treatment for high-risk patients with mixed dyslipidemias, and side effects, mainly hepatic and muscular disorders, are rare. However, when they do occur, muscular damage may be severe, leading to rhabdomyolysis (3)(4).
High-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy is a nondestructive and noninvasive technique that can provide complete structural analysis of a wide range of organic molecules in complex mixtures. NMR spectroscopy has been used for the detection of low-molecular-weight metabolites present in biological fluids as a result of changes in physiologic status, toxic insult, or disease processes (5). NMR provides quantitative information on the low-molecular-weight metabolites present in the specimen studied and allows the detection of unexpected constituents related to disease or tissue damage. Urinalysis by NMR spectroscopy has led to detailed investigation of the excretion pattern in various physiologic and pathologic situations, such as inborn errors of metabolism, organ transplantation, and renal damage produced by acute toxic exposure to drugs or other xenobiotics (6)(7).
In the present study we used 1H-NMR spectroscopy to explore noninvasively the reversible renal tubular dysfunction in a patient who developed rhabdomyolysis during treatment with combined therapy for hyperlipidemia and to detect the influence of retrieval condition on renal function. In addition, the results of the NMR study were correlated with conventional clinical chemistry methods.
The patient, a 75-year-old woman with severe combined hyperlipidemia, was admitted to the hospital with symptoms of bilateral lower extremity myalgia and weakness, which had appeared 2 weeks after treatment with cerivastatin (0.6 mg/day) plus gemfibrozil (600 mg twice a day) (8). Laboratory investigation showed increased muscle enzymes and urinary excretion of myoglobin, whereas serum creatinine was normal. The diagnosis of rhabdomyolysis was established, and the patient was treated with aggressive hydration and urine alkalinization. Symptoms and laboratory abnormalities gradually improved, and within 10 days the patient was discharged.
Blood and urine samples were collected on admission and every day during the patient’s hospitalization. An aliquot of each urine sample was stored at -70 °C for the NMR study. Serum and urine constituents were measured on an OLYMPUS 600 analyzer by standard procedures. The pH of the samples was 7.7 ± 0.2 for all samples except for the sample for day 8, which was 6.5. A standard formula was used to calculate the fractional excretion (FE). Urinary total protein was measured by the sulfosalicylic acid method. Urine protein composition (albumin, IgG, and 
1-microglobulin) was analyzed by immunonephelometry on a Behring BN100 nephelometer (Behring Diagnostics GmbH). Urine myoglobin concentrations were also measured by immunonephelometry, and ß2-microglobulin was determined on an AxSYM analyzer (Abbott Diagnostics).
For the NMR analysis, we added 50 µL of 2H2O containing sodium-3-trimethylsilyl-[2,2,3,3-2H4]-1-propionate (TSP) to 0.5 mL of crude urine as a chemical shift reference (
= 0.00). 1H-NMR measurements were made on a Bruker AMX400 spectrometer, operating at a field strength of 9.4 Tesla (400 MHz 1H frequency) at 22 °C. A continuous secondary irradiation field at the resonance frequency of water was applied to suppress the intense H2O signal. For each sample, 64 free induction decay (FID) measurements were collected into 16 384 computer points, with 90° pulses and spectral widths of 4800 Hz. The acquisition time per FID was 1.7 s, and a further delay of 6.3 s was added between pulses to permit full T1 relaxation. Resonances were assigned based on the chemical shifts relative to TSP, spin–spin coupling, and pH dependence of chemical shifts. Before Fourier transformation, an exponential line-broadening factor of 0.3 Hz was applied. Metabolite resonances were quantified by integration relative to that of TSP and expressed in mmol/mol of creatinine. NMR analysis of the urine of healthy individuals was used for comparison.
The excretion profiles of the low-molecular-weight metabolites in urine samples during the patient’s hospitalization, as detected by 1H-NMR spectroscopy, are illustrated in Fig. 1
, whereas the main quantitative alterations in the excreted metabolites are reported in Table 1
. The main changes observed in the NMR spectra the first 4 days of hospitalization were complete suppression of hippurate excretion and decreased excretion of citrate, indicative of a renal tubular malfunction, and increased excretion of dimethylamine (DMA) and trimethylamine N-oxide (TMAO), which are considered as markers for papillary dysfunction (6)(7). These findings indicate the presence of a generalized disturbance of the tubules that also affected the renal papillae, despite serum creatinine values within the reference interval. It is noticeable that there is no marker in conventional clinical chemistry to diagnose damage in renal papillae and that the concentration of TMAO, a specific marker for this part of the kidney, as has been revealed by NMR spectroscopy, was higher during the early stage of the disturbance (i.e., in the patient’s urine sample on admission) and then decreased to reference values after myoglobin excretion decreased. The tubular handling of glucose, amino acids, and lactate was not affected except for a decrease in glycine excretion. In addition, in the NMR spectrum of urine, significant concentrations of creatine were detected the first 3 days of hospitalization.
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An increase in hippurate excretion and a decrease in the excretion of DMA, TMAO, and creatine followed the progressive improvement in muscle function, as observed by the decrease in serum muscle enzymes (Table 1
). However, in the NMR spectrum, citrate and glycine excretion was further reduced along with a persistent increase in the FE of uric acid and potassium, findings that are indicative of the presence of a residual renal disturbance. A peak in the acetate resonance appeared only in a single urine sample, on the 8th day of hospitalization.
As is also shown in Table 1
, the tubular damage detected by NMR spectroscopy was supported by additional findings determined by various conventional clinical chemistry methods. On admission, hypouricemia with inappropriate uricosuria (FE of uric acid >10%), renal phosphate wasting (FE of phosphate >20%), inappropriate kaliuria (FE of potassium >9%), and serum potassium concentrations near the lower limit of the reference interval were found (9)(10). Although the hypouricemia and uricosuria persisted during the patient’s hospitalization, the inappropriate kaliuria and phosphaturia were rapidly resolved. However, there was no evidence of glucosuria or bicarbonuria, and arterial blood gases were repeatedly within the appropriate reference intervals.
Urinary excretion of 1- and ß2-microglobulin, specific markers for tubular damage, was increased the first 3 days of hospitalization and then started to decrease toward reference values. This decrease followed alterations in myoglobin excretion. Additionally, small quantities of albumin and IgG were detected in the urine the first 5 days. The slightly increased FEs for uric acid and potassium observed the 8th day of hospitalization are in agreement with the residual disturbance in the excretion of citrate and glycine observed by the NMR spectroscopy, as mentioned above.
Two months later, the patient’s laboratory findings were within reference values, with complete restoration of the NMR profile.
The NMR analysis presented in this study followed the progress of the tubular disturbance and revealed detailed information on the localization of the damage. Rhabdomyolysis is usually followed by renal injury, which has been related to renal vasoconstriction, intraluminal cast formation, and direct heme protein-induced cytotoxicity (1). However, in the absence of acute renal failure, there are no data on the development of reversible renal tubular damage in these patients. Our study clearly showed that rhabdomyolysis is followed by a reversible generalized tubular disturbance along with papillary dysfunction, as evidenced by disturbances in the excretion of low-molecular-weight metabolites (hippurate, citrate, DMA, and TMAO), as well as by renal wasting of uric acid, phosphate, and potassium and increased excretion of 1- and ß2-microglobulin. Most of these abnormalities improved during the patient’s hospitalization and followed the progressive decrease in myoglobin concentrations, suggesting that the presence of high myoglobin concentrations in the renal tubules was mainly responsible for the tubular damage.
The slightly increased urinary excretion of albumin and IgG, specific markers for glomerular damage, was not indicative of a glomerular dysfunction; it is known that tubular dysfunction is accompanied by a small increase in the excretion of these proteins, mainly of albumin (11).
In previous studies, NMR urinalysis in humans and experimental animals showed that damage to tubules and papillae leads to specific changes in the composition of urine (5)(6)(7)(12)(13). The main alterations observed in our study were decreased excretion of hippurate and citrate and increased excretion of DMA and TMAO. Hypocitraturia has been attributed previously to nephrotoxin-induced alterations in tubular acid–base status together with effects on the Krebs cycle (6). Hippurate, synthesized in the kidney and liver from glycine and benzoic acid, is secreted by the renal tubular cells and continually excreted in the urine. A significant decrease of this metabolite in urine may be indicative of a metabolic alteration and, even more importantly, of the efficiency of tubular secretion (14). TMAO has been shown to be confined to the inner medulla and has been suggested to play a role in the maintenance of intracellular osmotic balance in this region. Experimental studies have shown that increased urinary concentrations of TMAO and DMA are indicative of damage to the renal papillae. Interestingly, after renal transplantation the process of rejection has been significantly associated with increased urinary TMAO concentrations (5)(6)(7).
In our patient, the tubular handling of glucose, amino acids, and lactate was unaffected, indicating that the damage in the renal tubules had not compromised the S1 and S2 parts of the proximal tubules, where glucose and amino acids are mainly reabsorbed. Excretion of these metabolites in urine is noticed only in cases of severe proximal tubular damage (6)(12). It is of interest that a decrease in glycine excretion was also noticed. The underlying mechanism of this abnormality is not well known, but it may be related to damage to the S3 region of the proximal tubules, where glycine is handled (14). Finally, the increased urinary excretion of creatine may result from loss of creatine-containing tissues, such as muscles, and may represent an underlying defect in energy metabolism (1)(15).
Our findings show that rhabdomyolysis-induced myoglobinuria is followed by rather mild and rapidly reversible generalized tubular damage, despite the absence of renal failure. It is worth mentioning that it is possible to detect, in a single NMR spectrum, various compounds excreted in urine, providing detailed information on renal damage, even for slight residual disturbances, that is compatible with the conventional clinical chemistry methods, for which multiple methodologies and instruments are needed.
References
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