Carnitine is a naturally occurring substance in the human body required for energy metabolism at the cellular level. It has been shown to have a role in transporting fatty acids into mitochondria to help produce energy and in removing toxic waste from the cells. Studies indicate that more than 70% of the carnitine present in the plasma of a hemodialysis patient is removed during a single dialysis session. This high percentage carnitine loss by a patient during a hemodialysis session is thought to be attributable to the compound's relatively small molecular weight, high water solubility, and poor protein binding. It is believed that carnitine levels are further diminished in end stage renal disease (“ESRD”) patients by reduced renal synthesis and reduced intake of meat and dairy foods. Thus, the carnitine levels of dialysis patients, and end stage renal disease patients undergoing dialysis in particular, generally decrease steadily over time.
The depletion of carnitine levels in tissue is a noted long-term consequence of repeated losses of carnitine from plasma. Thus, carnitine depletion in tissue is experienced by patients that undergo successive dialysis sessions over a prolonged period of time. Tissue carnitine depletion is undesirable because it is associated as a possible causal factor for many health complications associated with dialysis, including cardiomyopathy, arrhythmias, muscle weakness, and general fatigue.
As described in U.S. Pat. Nos. 6,335,369 and 6,429,230 to Cavazza (henceforth, “the Cavazza patents”), the administration of levocarnitine (or L-carnitine) may be beneficially used to treat carnitine deficiency in ESRD patients (also known as “chronic uremic patients”) undergoing regular dialysis. According to the Cavazza patents, ESRD patients undergoing periodical dialysis can be administered levocarnitine or one of its salts to prevent or treat carnitine deficiency. This is taught to be accomplished by, among other things, administering an effective dose of L-carnitine intravenously via a venous return line after each dialysis session of the ESRD patient. The Cavazza patents teach that initiation of such L-carnitine injection therapy to treat carnitine deficiency may be prompted by patients demonstrating pre-dialysis plasma carnitine concentrations that are below normal (normal carnitine concentration levels are 40–50 μmol/L). Such intravenous administration of L-carnitine to ESRD patients undergoing dialysis treatments is taught to result in increased plasma carnitine concentrations. Additionally, carnitine deficiency in ESRD patients who are undergoing dialysis can be prevented by the intravenous administration of levocarnitine prompted by carnitine concentration levels in plasma that are trending toward carnitine deficiency. The methods for the prevention of treatment of carnitine deficiency as taught by the Cavazza patents thereby make it possible to correct for the loss of plasma carnitine which otherwise could take place during a dialysis session.
In order to manage the therapeutic delivery of L-carnitine to dialysis patients, it would be beneficial for physicians to have an accurate and reliable, yet fast and low cost, method to monitor the level of plasma carnitine in patients before a given dialysis session, after a given dialysis session, and/or during a given dialysis session. The current techniques available for measuring carnitine levels in a patient's plasma include enzymatic assays, high-pressure liquid chromatography (“HPLC”), and tandem mass spectrometry (“MS/MS”). These techniques for quantifying plasma carnitine levels, however, do not generally provide the high throughput, low cost, and high sensitivity and accuracy analysis necessary for implementation on a large-scale basis to manage levocarnitine replacement therapies for patients undergoing dialysis.
Each of the contemporary techniques for measuring carnitine levels in patients suffers from distinct disadvantages. Enzymatic assays are highly sensitive, but are extremely time consuming to run and, therefore, are not readily adaptable to large-scale use by dialysis clinics. Furthermore, enzymatic assays can only provide quantification of free carnitine (L-carnitine) and total carnitine levels in a plasma sample and do not provide results for individual acylcarnitines, such as long chain carnitines, which are useful markers of the metabolic status of ESRD patients undergoing dialysis for selecting appropriate therapies. Furthermore, the determination of total carnitine levels using enzymatic assays must be carried out separately from the determination of free carnitine levels due to the necessary use of chemical hydrolysis prior to quantification.
HPLC methods involving specific pre-column derivatization and fluorescence detection have been demonstrated to be sufficiently sensitive to allow the determination of levels for several individual acylcarnitines. However, HPLC methods remain expensive and very time consuming, in large part because the sample preparation require elaborate, multi-step procedures. Furthermore, the determination of long chain and very long chain acylcarnitine levels cannot be carried out by HPLC. HPLC further suffers from the fact that the quantification of total carnitine levels requires a separate analytical run including chemical hydrolysis of the sample.
Notably, both the HPLC and enzymatic assays methods that can be used for accurate and reliable determination of carnitine levels require biological samples that have been kept frozen during storage and shipment following collection. Understandably, this factor makes remote laboratory utilization of these procedures difficult.
With regard to MS/MS quantification of carnitine levels, electrospray ionization MS/MS has been used to identify abnormal carnitine profiles in infants from blood samples. In particular, U.S. Pat. No. 6,258,605 to Chace (“the '605 patent”) discloses a method for genetic screening of infants using tandem mass spectrometry (“MS/MS”). According to the '605 patent, blood is taken from an infant patient and spotted in small amounts on a piece of filter paper where it is dried and sent to a lab. The blood is then reconstituted at the lab and run through an electrospray ionization mass spectrometer with an internal standard to identify potentially abnormal metabolic profiles of amino acids and acylcarnitines. By collecting dried blood samples on filter paper, the method taught by the '605 patent allows for inexpensive, un-refrigerated transfer of blood samples from the doctor to a remote lab for analysis. This in turn permits a single, remotely located mass spectrometer to be used to handle a large volume of samples, thereby taking advantage of economies of scale.
Although electrospray ionization MS/MS of dried blood samples as taught by the '605 patent is a reliable, low cost and high throughput mechanism for identifying abnormal metabolic profiles of acylcarnitines in infants, it is not a suitable solution for obtaining accurate quantifications of plasma carnitine levels in carnitine deficient patients for several reasons. First, blood can be a poor indicator of tissue carnitine levels because the intracellular concentration of L-carnitine in red blood cells does not equilibrate with the plasma concentration (the membranes of the erythrocytes being impermeable to L-carnitine). Only the carnitine present in a patient's plasma can be dialyzed during a given dialysis session while the carnitine concentration within the red blood cells is not directly affected by dialysis. Furthermore, since red blood cells do not have mitochondria, carnitine cannot be utilized therein for the β-oxidation of long-chain fatty acids. Therefore, the intracellular concentration of carnitine present in red blood cells may in fact skew any analyses for carnitine deficiency. Preparation of dried blood samples for introduction into the mass spectrometer as taught in the '605 patent would necessarily release a large portion of carnitine from the red blood cells and thereby overstate the determination of free and total carnitine levels.
In addition, the derivation steps employed during sample preparation for many MS/MS analysis methods are known to cause hydrolysis of acylcarnitines into free carnitine, as has been reported by Johnson et al. (1999) among others. This hydrolysis of acylcarnitines can cause over-reporting of free carnitine. Unfortunately, such over-reporting of free carnitine is clinically significant if the method were to be used to manage carnitine replacement therapy for dialysis patients. Prior solutions to this problem entail running an MS/MS assay twice on a given sample, once without derivatization to measure free carnitine levels and a second time with derivatization to measure total carnitine levels. This, however, increases sample analysis costs and therefore loses a significant benefit of MS/MS methods. Furthermore, while MS/MS can be employed without a derivatization step, removal of this step causes the sensitivity of the assay to decrease over ten fold. This marked decrease in sensitivity is problematic in the quantification of carnitine deficiency because the screened samples taken from carnitine deficient patients undergoing dialysis are necessarily expected to have low levels of carnitine present.
A further complication to the adaptation of MS/MS to quantifying carnitine levels is that glutamic acid is believed to interfere with the analysis of acetylcarnitine concentrations. Tandem mass spectrometry is capable of distinguishing molecular ions of the same mass that also have different product ions. Tandem mass spectrometry, however, cannot distinguish between molecular masses having the same product ions and molecular ions. The di-butyl ester of the protonated from of glutamic acid shares the same molecular ion molecular mass (260 m/z) and product ion molecular mass (85 m/z) as the butyl ester of acetylcarnitine. Since glutamic acid in physiological concentrations is much more abundant than acetylcarnitine, the protonated form glutamic acid can be present in sufficient amounts that can interfere with the MS/MS signal at 260 m/z which is associated with the detection of acetylcarnitine. Therefore any utilization of MS/MS for analysis of carnitine concentrations must account for this problem.
Therefore, there remains a need in the art for an improved method for diagnosing carnitine deficiency in patients and quantifying the level of carnitine deficiency such that carnitine concentrations can be easily and accurately tracked within a given patient over time. Also, there remains a need in the art for a high throughput, low cost, and high accuracy method for quantifying the level of carnitine deficiency in a patient such that the method can be used frequently to monitor the level of carnitine deficiency and manage appropriate therapies to treat carnitine deficiency.