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Clinical Chemistry 43: 121-128, 1997;
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(Clinical Chemistry. 1997;43:121-128.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Measurement of strontium in serum, urine, bone, and soft tissues by Zeeman atomic absorption spectrometry

Patrick C. D'Haese, Glen F. Van Landeghem, Ludwig V. Lamberts, Vera A. Bekaert1, Iris Schrooten and Marc E. De Broea

Department of Nephrology–Hypertension, University of Antwerp, Antwerp, Belgium.
1 Department of Industrial Sciences and Technology, Catholic Polytechnic Institute Antwerp, Antwerp, Belgium.
a Address correspondence to this author at: University of Antwerp, Department of Nephrology–Hypertension, p/a University Hospital Antwerp, Wilrijkstr. 10, B-2650 Edegem/Antwerpen, Belgium. Fax +32/3/829-0100; e-mail debroe{at}uia.ua.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
To study the possible accumulation of Sr in chronic renal failure patients, methods were developed for the determination of the element in serum, urine, bone, and soft tissues by using Zeeman atomic absorption spectrometry. Serum samples were diluted 1:4 with a Triton X-100–HNO3 mixture, whereas urine samples were diluted 1:20 with HNO3. Bone samples were digested with concentrated HNO3 in stoppered polytetrafluoroethylene (Teflon®) tubes, whereas soft tissues were dissolved in a tetramethylammonium hydroxide solution in water. For serum and urine we used matrix-matched calibration curves, whereas bone and tissue samples were measured against aqueous calibrators. Atomization was performed from the wall of pyrolytically coated graphite tubes for all of the matrices under study. Both inter- and intraassay CVs were <6% (n = 12, n = 10, respectively), and the recovery of added analyte was close to 100% for all of the biological matrices under study. Detection limits were 1.2 µg/L (serum), 0.3 µg/L (urine), 0.4 µg/g (bone), and 2.2 ng/g (soft tissues), whereas the sensitivity determined by the slope of the calibration curve, i.e., the amount of Sr producing a 0.0044 integrated absorbance change in signal, was 2.4 pg, 2.4 pg, 3.9 pg, and 2.6 pg for these matrices respectively. We conclude that the present methods are precise and accurate and easily applicable for both routine use and research investigations. They will allow us to study the metabolism of the element in chronic renal failure patients and shed some light on the association that was recently noted between increased bone Sr concentrations and the development of osteomalacia in these individuals.


Key Words: indexing terms: dialysis • chronic renal failure • renal osteodystrophy • osteomalacia


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Sr, a member of the earth alkaline elements, resembles to a great extent Ca. Like the latter element, >99% of the total Sr body burden is localized in bone. Whether the element plays an essential role in bone metabolism is not yet fully understood. In rats the oral administration of low doses of Sr may stimulate bone formation. When given at high doses, however, Sr may induce a defective bone mineralization expressed by a decreased bone mineral density and decreased size of bone apatite (1)(2). In this context our recent findings of increased bone Sr concentrations in dialysis patients with osteomalacia (3) are of particular interest. At present it is not clear if in these individuals Sr has played either a primary or contributive role in the development of this particular type of renal osteodystrophy, nor is it clear if the increased concentrations occurred secondary to the presence of the bone disorder. Also, no unequivocal data are available in the current literature as to what extent chronic renal failure or the dialysis treatment per se leads to accumulation of the element. In the latter population, the element's ability to interfere with Ca homeostasis and vitamin D metabolism might be of particular interest, since in dialysis patients both of these processes are disturbed (4)(5).

In 1986, Canavese et al. (6) stated that a better identification of the causal factors of the Sr imbalance in end-stage renal failure patients and information on the biological nature of Sr in biological fluids and tissues are necessary to better understand the effects of the element on its target tissue, more particularly the bone. Also, the element has recently been proposed as a potential therapeutic agent in the prevention and treatment of osteopenic bone lesions (7). This implies that biological monitoring of Sr may become important in the near future.

The beneficial as well as deleterious effects of Sr on bone have been investigated in several experimental and some human studies (1)(2)(8)(9). In the great majority of studies on the biological effects of Sr, however, data on Sr concentrations are lacking. Remarkably, this goes along with a scarcity of reports describing analytical methods that are sensitive and accurate enough to determine the element in biological fluids, bone, and tissues.

Techniques applicable for analyzing Sr are flame atomic emission spectroscopy with a filament vaporizer (10), inductively coupled plasma atomic emission spectrometry (11), direct-current plasma echelle spectrometry (12), neutron activation analysis (13), and x-ray fluorescence (14); these techniques are not currently available in the clinical laboratory. Here analysts mostly have to rely on atomic absorption spectrometry (AAS), either based on flame (FAAS) or electrothermal atomization (ETAAS).1 FAAS in general is not sensitive enough to directly determine the element at the relatively low (ppb-ppm) concentration ranges at which it occurs in biological fluids and tissues. Moreover, in the analysis of biological material, FAAS-based methods are known to be subject to several chemical interferences. In view of its potential analytical performance, ETAAS is at present the method of choice to determine Sr in biological samples. However, even in the presence of the latter technique, several difficulties have been reported in the analysis of serum and urine (15). Furthermore, data in the literature related to the ETAAS determination of Sr in more complex matrices such as bone are extremely scarce and to the best of our knowledge do not exist for the measurement of the element's low concentrations in soft tissues.

In view of the above and to allow us to study the accumulation/distribution of Sr in dialysis patients and to investigate whether in these subjects the presence of particular types of renal osteodystrophy are accompanied by an increased/decreased Sr concentration, Zeeman ETAAS-based analytical methods for the element's determination in serum and urine were further optimized and were developed for bone and soft tissue analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
apparatus
Analyses were performed with a Zeeman 3030 AAS equipped with an HGA-600 graphite furnace, an AS-60 autosampler, and an Anadex DP-9500B silent scribe printer, all from Perkin-Elmer, Norwalk, CT. The light source was a hollow cathode lamp, which was used at 25 mA at 460.7 nm. Instrument settings for the various biological matrices are presented in Table 1 . Atomization was performed from the wall of pyrolytically coated graphite tubes.


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  		Table 1. ETAAS determination of Sr in serum, urine, bone, and tissues.

materials
Materials used were either 5- or 10-mL polystyrene tubes, 50-mL polypropylene volumetric flasks (Brand, Wertheim, Germany), Teflon tubes (VEL, Leuven, Belgium) equipped with loosely fitting Teflon caps, and polystyrene sample cups (Biolab, Limal, Belgium). All automated pipettes used were "Finnpipettes" (Labsystems, Helsinki, Finland) equipped with disposable polypropylene pipette tips. The use of glassware was avoided since this material might be contaminated with Sr (16).

reagents
Concentrated HNO3 (Suprapur 441; Merck, Darmstadt, Germany) and concentrated Triton X-100 (BDH Chemicals, Poole, UK) were used to prepare the sample dilution reagents. Soft tissues were dissolved in a 100 g/L tetramethylammonium hydroxide (TMAH) (Janssen Chimica, Geel, Belgium) solution in water, whereas for bone digestion concentrated HNO3 (Suprapur, Merck) was used. EDTA was from Merck (Art 8418, "Titriplex III"). A 1 g/L stock calibrator of SrCO3 in 0.3 mol/L HNO3 (J.T. Baker, Philipsburg, NJ) was used to prepare intermediate and working calibrators.

samples and sample taking
Serum, urine, bone, and tissue samples were taken and stored as described previously (17). Optimization of the methodology for Sr in serum was done with samples that were taken from both dialysis patients and individuals with normal renal function. Bone samples were transiliac biopsies from dialysis patients that were taken in the frame of previous studies of our group on the diagnosis and prevalence of particular types of renal osteodystrophy (18)(19). For method development for Sr in urine, samples were taken from healthy humans, whereas for soft tissues rat specimens were used.

sample preparation
Serum.
The serum samples were diluted fourfold in a 0.5 mL/L Triton X-100–1 mL/L HNO3 solution.

Urine.
Urine samples were diluted 20-fold in a 2 mL/L HNO3 solution.

Bone.
Wet weighed bone biopsy samples (10–500 mg) were quantitatively transferred to 10-mL Teflon tubes to which either 1 mL or 2 mL (according to the sample weight) of concentrated HNO3 was added. Tubes were closed by means of loosely fitting Teflon caps to allow digestion at atmospheric pressure. These were placed in an oven for 3 to 4 h at 80–90 °C until a clear digest was obtained. The digestion liquid was then quantitatively transferred to either 25-mL or 50-mL polypropylene volumetric flasks and adjusted to the appropriate volume with deionized/reverse osmosis-treated water. This solution was then transferred to two 10-mL polystyrene tubes and stored at -20 °C; the digestion liquids were diluted 40-fold in an aqueous 0.5 mL/L Triton X-100–0.5 mL/L HNO3 solution before analysis.

Soft tissues.
Wet weighed soft tissue samples (100–300 mg) were dissolved in 10-mL stoppered graduated polystyrene tubes after the addition of 1 to 3 mL (according to sample weight; ± 1 mL/100 mg sample) of a 100 g/L TMAH solution in water. Caps of tubes were perforated with an 18-gauge syringe needle to allow evaporation and make dissolution at atmospheric pressure possible. Solubilization was performed for at least 12 h (overnight) at 60 °C. Samples were regularly mixed by means of a vortex-type mixer to accelerate the dissolution process. After completion of dissolution, samples were, according to the expected concentration or sample weight, adjusted to the appropriate volume (1:2 up to 1:5 dilution) with an aqueous 20 g/L TMAH–2 g/L EDTA solution in water.

calibration
From a 1 g/L stock calibrator solution of SrCO3 in 0.3 mol/L HNO3, an intermediate calibrator of 1 mg/L was prepared by diluting 50 µL of the stock calibrator in 50 mL of a 20 mL/L HNO3 solution. From this intermediate calibrator, working calibrators with appropriate concentrations were prepared in either aqueous, matrix-matched, or sample solutions for comparison of the direct calibration, matrix-matched calibration, and standard addition technique. Comparison of these yielded the following calibration procedures:

Serum.
To 100 µL of a serum pool with relatively low Sr content, 100 µL of working calibrators of 0, 12.5, 25.0, 50.0, and 100.0 µg/L in 0.5 mL/L Triton X-100–1 mL/L HNO3 and 200 µL of the blank 0.5 mL/L Triton X-100–1 mL/L HNO3 solution were added, which yielded a matrix-matched calibration curve that contained Sr calibrators of 0, 3.13, 6.25, 12.5, and 25.0 µg/L, respectively.

Urine.
To 100 µL of a urine pool with relatively low Sr content, 100 µL of working calibrators of 0, 62.5, 125.0, and 250.0 µg/L in 2 mL/L HNO3 and 1800 µL of the blank 2 mL/L HNO3 solution were added. A matrix-matched calibration curve containing Sr calibrators of 0, 3.13, 6.25, and 12.5 µg/L, respectively, was obtained.

Bone.
For bone an aqueous calibration curve was constructed by preparing working Sr calibrators of 0, 3.13, 6.25, 12.5, and 25.0 µg/L in 0.5 mL/L Triton X-100–0.5 mL/L HNO3.

Soft tissues.
Here, an aqueous calibration curve was prepared by diluting the intermediate calibrator in a 20 g/L TMAH–2 g/L EDTA solution so that working calibrators of 0, 3.13, 6.25, and 12.5 µg/L were obtained.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
choice of graphite tube
In the presence of uncoated graphite tubes, atomization from the wall yielded no signal (Fig. 1 ). Insertion of a L'vov platform did not result in any improvement, either in the presence of an uncoated tube or when a pyrolytically coated graphite tube was used or when the atomization temperature was increased to 2700 °C. A possible explanation for this lies in the fact that optimum atomization temperatures with the L'vov platform are typically somewhat higher than those for direct atomization off the tube wall. In the determination of the relatively involatile Sr, the required temperature is probably not achieved in the presence of the platform.



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  		Figure 1. Influence of the choice of graphite tube on atomization signal of Sr.

Acceptable atomization signals were only obtained after atomization from the wall in the presence of pyrolytically coated graphite tubes. For Sr, atomization from the wall of pyrolytically coated graphite tubes has also been recommended by Slavin et al. (20), suggesting that the element, like the other earth alkaline metals, gets intercalated in the graphite of uncoated tubes, resulting in poor atomization signals. Others have used metal microtube atomizers made from a small inner-diameter tungsten tubing to improve the sensitivity and detection limits of the ETAAS determination and that of other relatively involatile elements (21).

In view of the nonideal peak shape with even pyrolytically coated graphite tubes, integrated absorbance (Ai) measurements were preferred to peak height despite the lower sensitivity in the latter measurement mode. However, because of the better precision that was noted when the Ai signals were used, measurement in the peak area mode did not result in poorer detection limits.

instrument settings
A typical char-atomization curve in the presence of pyrolytically coated graphite tubes is presented in Fig. 2 , showing Sr to be lost at temperatures >1500 °C, whereas the plateau of maximal absorbance is reached at atomization temperatures >2200 °C. The internal gas flow had an important influence on the sensitivity (Fig. 3 ). In contrast to others using nitrogen (15), we used argon as purge gas, which recently was shown to have a pronounced effect on both the absorbance signal (50% increase) and the lifetime of the graphite tube (16). Balancing the loss in sensitivity against the expected concentration in the biological matrices under study, reproducibility, and tube lifetime, a miniflow of 50 mL/min instead of gas-stop was used during atomization for the determination of Sr in serum, bone, and urine. In view of the extremely low Sr concentrations noted in soft tissues for the analysis of these samples, the gas-flow during atomization was reduced to 10 mL/min to increase the sensitivity.



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  		Figure 2. Typical char-atomization curves for ETAAS determination of Sr.

Curves did not differ significantly for the various biological matrices.



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  		Figure 3. Influence of internal argon gas flow on sensitivity of Sr determination.

We used a slit width of 0.2 nm, which had a significant effect on the linearity of the calibration curve as compared with when a 0.7-nm slit width was used (Fig. 4 ). An overview of the instrument parameters is provided in Table 1Up .



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  		Figure 4. Influence of slit width on curve linearity range in the determination of Sr.

sample preparation
For the determination of Sr in serum and bone digestion liquids, the addition of Triton X-100 resulted in a better reproducibility without sensitivity loss. Perhaps this was due to a better sample deposition into the graphite furnace, which in turn was accompanied by a more regular drying of the sample. The addition of Triton X-100 was not necessary for the ETAAS analysis of urine samples. HNO3 acid had a beneficial effect on the signal stability; however, it was not necessary in the analysis of soft tissue samples.

Sr in bone was determined after simple decomposition in HNO3 under atmospheric pressure in loosely stoppered Teflon test tubes. Here, a clear digest is already obtained after a 3–4-h digestion at 90 °C. After diluting the digestion liquid to the appropriate concentration range, Sr could directly be measured in the diluted digestion liquid. With the proposed method, the use of expensive digestion bombs or decomposition instrumentation is not necessary and a considerable number of samples can be analyzed in a day. In our laboratory the procedure has also been used successfully for the determination of a series of other trace metals (17)(22)(23). Hence it allows multielement determination in one bone biopsy, which in view of the rather complex sampling procedures of these specimens must be considered an important advantage.

In view of the ultralow concentration at which Sr occurs in soft tissues as compared with bone, HNO3 digestion could not be used in the analysis of the former specimen. Indeed, in the presence of such low concentrations, further dilution of the digestion liquid is limited, yielding an unacceptable high HNO3 concentration in the final solution, which in turn would have a deleterious effect on the lifetime of the graphite tube and alter the absorption signal. To circumvent this problem we opted for a solubilization procedure in a 100 g/L TMAH solution wherein the element could either be measured directly or after a limited dilution in TMAH-EDTA. Since dissolution was performed in graduated polystyrene tubes, quantitative volume adjustment could be performed in the same recipient by which an additional transfer step to volumetric flasks was omitted. Here the addition of EDTA resulted in a significant (±30%) increase in sensitivity. Perhaps this is due to the ability of EDTA to form a chelate with Sr, which results in a more adequate atomization of the element by preventing it from forming either stable or volatile compounds with cations present in the sample matrix. The use of TMAH has previously been used successfully in our laboratory for the determination of Pb and Cd in soft tissue samples also (data not published). Stevens (24) used the latter reagent for the determination of Al in soft tissues. However, with TMAH, bone samples cannot be solubilized, making the latter method unsuitable for these specimens.

calibration
The linearity of the calibration curves was tested by calculating the correlation coefficient, which should be >0.99 in combination with the determination of the y-residuals (i.e., difference between the experimental y-values and the fitted y-values) representing the random experimental errors over the concentration range (see inset, Fig. 4Up ). Calibration curves were linear up to a Sr concentration of 200.0 µg/L, provided a slit width of 0.2 nm was used. Here a 200 µg/L Sr concentration corresponded with 2.5 Ai units. This corresponds with serum and urine Sr concentrations of 6.2–800 µg/L and 31.2–4000 µg/L, respectively. Calibration in the lower concentration range is depicted in the inset.

Calibration curves prepared for the biological matrices under study and comparison with aqueous calibrators are presented in Fig. 5 . Confidence interval analysis on the difference between slopes of linear regression curves (25) indicated a significant difference between slopes of aqueous and urine-matched standard-addition curves. No significant differences were noted between aqueous calibration curves and standard-addition curves prepared for the other matrices under study. For each of the matrices under study, the slopes of the standard-addition curves prepared from samples of different subjects did not differ significantly from each other. Hence, the cumbersome standard-addition technique was not required for each of the matrices under study. Direct standardization with aqueous calibration curves could be used for the determination of Sr in bone and soft tissues. Because for serum a better analytical performance was obtained with matrix-matched calibration curves than with aqueous calibration curves, as for urine, we used the former calibration technique for serum also.



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  		Figure 5. Comparison of slopes of serum-, urine-, bone digestion liquid-, and tissue solubilization liquid-matched calibration curves (lower panels) vs aqueous calibration curves (upper panels).

Comparison of slopes of serum-, urine-, and bone digestion liquid-matched calibration curves prepared from samples of three different subjects (S.1–3) and of slopes of tissue solubilization liquid-matched calibration curves prepared from heart ({blacksquare}), spleen ({square}), and muscle (•) tissue (lower panels).

analytical performance
Data on the analytical performance of the Sr determination in the various biological matrices as evaluated in terms of within- and between-run CVs, recovery of added analyte, recovery of digestion, sensitivity (determined by the characteristic mass, i.e., the amount of Sr yielding 0.0044 Ai units), limit of detection (mean blank + 3 SD), and limit of quantification (mean blank + 10 SD) are presented in Tables 2 and 3.

For each of the matrices under study the within-run precision (repeatability) was assessed by determination of the Sr concentration in three samples containing Sr from the lower up to the higher concentration range (see Tables 2 and 3 ). Five dilutions were prepared per specimen, which were analyzed in two replicates within one run. For between-run precision (reproducibility), we determined weekly over a 21-day period in two replicates the Sr content in two dilutions of three samples of each of the biological matrices under study, covering a low to high concentration range.


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  		Table 2. ETAAS determination of Sr in serum and urine—analytical performance.


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  		Table 3. ETAAS determination of Sr in bone and soft tissues—analytical performance.

At present, no Certified Reference Material (CRM), external quality-control schemes, or reference quality methods are available for the determination of Sr in serum or urine. Accuracy of the bone and tissue analyses was checked by means of the Community Bureau of Reference (BCR) CRM no. 278 (mussel tissue) (Table 4 ). To check the accuracy of the proposed method for tissue analysis in the ultralow concentration range, we used the Standard Reference Material (SRM) no. 1577 (bovine liver tissue material) (Table 5 ). Data indicate the proposed method to be accurate.


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  		Table 4. Accuracy of methods for determination of Sr in bone and soft tissues.


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  		Table 5. Accuracy of method for determination of ultralow concentrations of Sr in soft tissues.

clinical relevance of determining sr in blood and tissues
Mean Sr concentrations assessed in serum of 25 subjects with normal renal function were 30.9 ± 11.7 µg/L. These correspond well with the lower concentration range of the data reported in the literature (26). Preliminary data of our group obtained in an ongoing worldwide multicenter study indicate that, as previously noted for other elements, e.g., silicon (27), serum Sr concentrations in dialysis patients may differ from country to country and center to center, varying between values noted in individuals with normal renal function up to concentrations 10–20 times higher. This is in agreement with the scarce data in the literature on Sr concentrations in dialysis patients, which also show a wide variation in the serum Sr concentrations (6)(28)(29). Canavese et al. (6) reported values in patients on renal dialysis treatment of 56.1 ± 17.4 µg/L, whereas Wilhelm et al. (28) found predialysis serum Sr concentrations of 185.3 µg/L ± 34.8 µg/L, which increased up to 330.0 ± 91.6 µg/L at the end of dialysis. These latter figures indicate that the Sr content of the dialysis fluid plays an important role in the accumulation of the element in the dialysis population. As already established for other elements (27), further investigations are required to determine, besides contamination of the dialysis fluid, to what extent renal failure itself and (or) diet also determine serum Sr concentrations in dialysis patients.

Our recent observation of increased Sr concentrations in bone of dialysis patients with osteomalacia is intriguing (3). At present it is not yet clear whether Sr has played a causative role in the development of this type of bone disease, or if accumulation of the element occurred secondary to the presence of osteomalacia. In this context it is worth mentioning that in preliminary studies in a chronic renal failure rat model, osteomalacia could be induced after Sr loading (30). Others have also in both experimental and human studies demonstrated the element to interfere with bone metabolism (1)(2)(8). On the other hand, Sr has a potential therapeutic value in the prevention and treatment of osteopenic disorders (7).

At present it is not yet clear to what extent serum Sr concentrations reflect the element's body burden, i.e., bone Sr concentrations or potential toxicity. In view of the above observations, validation of a serum Sr determination in the monitoring and diagnosis of Sr overload/deficiency and treatment follow-up might become important.

The accurate determination of Sr gains in interest in view of the element's potential to mimic Ca absorption. Indeed, measurement of stable Sr instead of using the 45Ca isotope has recently been found to be a valuable safe alternative for Ca absorption tests (31)(32). The latter issue might become of particular interest in dialysis patients, who are known to be prone to a disturbed Ca absorption mechanism.

The determination of Sr in urine will be a useful tool in both experimental and human studies to (a) investigate the interactions of the element with, e.g., Ca; (b) get a better insight into the renal clearance; and (c) define a possible role for impaired renal function in the accumulation of the element in chronic renal failure patients.

The methodology for the determination of Sr in tissues will be helpful to get a better insight into the element's metabolism and tissue/cellular uptake and distribution. Also, the determination of Sr in organs and tissues might become of interest in view of the potential interaction of the element with, e.g., Ca, which in turn might influence parathyroid gland function and vitamin D synthesis, two biochemical processes that are altered in renal failure (4)(5).

The proposed methods for Sr determination in serum, urine, bone, and tissues are simple, sensitive, accurate, and precise. They allow routine determination as well as research investigations on the element at the lowest concentrations encountered in biological materials without the need for matrix modification. The STPF concept recommended for several trace metal analyses cannot be used for Sr because the element requires atomization from the wall of pyrolytically coated graphite tubes. We found that the use of TMAH offers a valuable alternative for the acid digestion of soft tissue samples, particularly when sample dilution of the digestion liquid is not possible because of the very low concentration at which the analyte occurs in the solid matrix under study. The described methods are useful for routine applications and are currently used successfully in both experimental and epidemiological studies on the potential role of Sr in the development of osteomalacia in chronic renal failure patients.


   Acknowledgments
 
We are indebted to E. Snelders for expert desk editing and to D. De Weerdt for his excellent drawings. Thanks are also due to M. Elseviers for her advice on statistics.


   Footnotes
 
1 Nonstandard abbreviations: FAAS, flame atomic absorption spectrometry; ETAAS, electrothermal atomic absorption spectrometry; TMAH, tetramethylammonium hydroxide; Ai, integrated absorbance; BCR, Community Bureau of Reference; SRM, Standard Reference Material; and CRM, Certified Reference Material.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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The following articles in journals at HighWire Press have cited this article:


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 Nephrol Dial TransplantHome page
G. B. Spasovski, A. R. J. Bervoets, G. J. S. Behets, N. Ivanovski, A. Sikole, G. Dams, M.-M. Couttenye, M. E. De Broe, and P. C. D'Haese
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[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
P. C. D'Haese, M.-M. Couttenye, L. V. Lamberts, M. M. Elseviers, W. G. Goodman, I. Schrooten, W. E. Cabrera, and M. E. De Broe
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[Abstract] [Full Text] [PDF]

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