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Letters |
1
Depts. of Biochem. and
3
Med. Informatics, Epidemiol., and Statistics, Univ. of Nijmegen, Nijmegen, The Netherlands,
2
Dept. of Clin. Chem.,
4
Divs. of Gen. Intern. Med. and
5
Nephrol., University Hosp. Nijmegen, Nijmegen, The Netherlands
a Address correspondence to this author at: P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
To the Editor:
About 30% of patients with insulin-dependent diabetes mellitus develop diabetic nephropathy. Since diabetic nephropathy contributes to a large extent to the high mortality of these patients, a risk marker for the development of this condition is desirable. Increase in Na+/Li+ countertransport across the red cell membrane has been suggested as such an early marker (1)(2)(3)(4), although this was not uniformly confirmed (5)(6)(7). In this study, the three main methods to load erythrocytes with Li+ were compared and tested for their measuring error and intrasubject variation of Vmax and K0.5 for Na+.
The "classic" LiCl loading [8, 9] is the most "physiological" and noninvasive method. However, the loading procedure takes 3 h, which precludes the use of this method as a standard procedure. The Li2CO3 loading as described by Elving et al. (6) has the advantage that it takes only 30 min to load the cells with Li+. The Li+ enters the cell via the HCO3-/Cl- exchanger as a LiCO3- ion in exchange for a Cl- ion, which explains the fast Li+ loading. This method has been reported to give plots to which Michaelis–Menten kinetics apply (10). The nystatin method was developed by Canessa et al. (11) because at 150 mmol/L Na+ (the highest concentration that can be used at an osmolarity of 300 mosmol/L), the extracellular binding site for Na+ is often not saturated. With nystatin, an antifungal drug that penetrates the plasma membrane, the intra- and extracellular osmolarity can be raised to 600 mosmol/L, so extracellular concentrations of Na+ up to 300 mmol/L can be used. Because of this, K0.5 values can in principle be measured more accurately than with the other methods. We found, however, that even at this high Na+ concentrations the Vmax of diabetic patients is often hardly reached.
Participants in this study were four male patients with insulin-dependent diabetes with diabetic nephropathy and four male healthy volunteers. The subjects had fasted overnight before a blood sample was taken.
The efflux media for the erythrocytes loaded with Li+ by using the LiCl or Li2CO3 methods contained 0–150 mmol/L NaCl, 150–0 mmol/L choline chloride, 1 mmol/L MgCl2, 10 mmol/L Tris-3-(N-morpholino)propanesulfonic acid (MOPS) buffer pH 7.4, 10 mmol/L glucose, and 0.1 mmol/L ouabain. Na+ concentrations were 0, 10, 20, 40, 60, 80, 100, 120, and 150 mmol/L. The sum of the concentrations of Na+ and choline was always 150 mmol/L. The efflux media for the erythrocytes loaded with Li+ by using the nystatin method contained 0–300 mmol/L NaCl and 300–0 mmol/L choline chloride; the rest of the medium was the same. Used Na+ concentrations were 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, and 300 mmol/L. Here the sum of the concentration of Na+ and choline was always 300 mmol/L. Li+ concentrations were measured by atomic absorption spectrometry (Model 4100; Perkin-Elmer, Norwalk, CT). The Vmax and K0.5 values were determined or extrapolated with the computer program Graphpad Inplot version 4.0 (Graphpad software, San Diego, CA). A rectangular hyperbola (binding isotherm) was fitted through the data. The following equation was used: V = A[Na+]/(B+[Na+]); A = Vmax, B = K0.5.
From each subject a blood sample was taken twice, with a time interval of 1 month. The data were analyzed and the Vmax and the K0.5 for Na+ were determined. R2 (coefficient of determination), a marker for the fit of the curve to Michaelis–Menten kinetics, was calculated. When the LiCl method was used, 25% of the R2 values were <0.7, which indicates a poor fit to Michaelis–Menten kinetics. There were no curves including Hill plots that fitted better. The nystatin method generated only one R2 value <0.7, and with the Li2CO3 method all R2 values were >0.7. It is clear that the data obtained with the Li2CO3 or nystatin method fit better to Michaelis–Menten kinetics than the data obtained with the LiCl method. This was a reason for us to continue with the methods involving Li2CO3 and nystatin.
Next, the CV of the measuring error of the data obtained with the
Li2CO3 or nystatin Li+ loading
methods was analyzed (with a paired t-test). At one day the
same sample of blood was analyzed twice (Table 1
). It was assumed that the difference was negligible between the
"month-to-month variation within one sample" and the measuring
error. It was further assumed that differences between two methods were
statistically independent from each other, both between subjects and
between months. For both methods the CV of the measuring error of the
K0.5 for Na+ did not significantly
differ from the measuring error of the Vmax
(nystatin P = 0.21; Li2CO3
P = 0.29). When the two methods are compared, there
seems to be no important difference in the CV of the measuring error of
the values for Vmax or
K0.5. For the Vmax the
difference between the values obtained with the nystatin and
Li2CO3 method was 0.6% (SD = 6.3,
P = 0.79) and for the K0.5 for
Na+ the difference was -5.3% (SD = 13.1,
P = 0.29).
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In addition, the intrasubject variation was examined (Table 1
). A blood
sample was taken three times, with intervals of 1 month. The samples
were analyzed with both the Li2CO3 and
nystatin Li+ loading methods. For both methods the
intrasubject variation for the K0.5 for
Na+ was large. Table 1
shows that for all methods the
Vmax is more constant than the
K0.5. For the nystatin, method comparison of the
values for the CVs of Vmax and
K0.5 for Na+ gives a difference of
22.7% (SD = 19.9, P = 0.015) and for the
Li2CO3 method the difference was 12.6%
(SD = 15.0, P = 0.05). From Table 1
it seems as if
the variation of the Vmax values obtained from
one individual is smaller when the nystatin method is used, although
this trend could not be supported statistically (P =
0.09).
Until recently the Na+/Li+ countertransport activity was measured as the Li+ efflux rate in medium containing 150 mmol/L Na+ after subtraction of the efflux rate in Na+-free medium. LiCl was used to load erythrocytes. As a discriminatory value, 400 µmol Li+/[hL red blood cells (RBC)] was used. Subjects with a countertransport above this value were described as at risk. The values we found for the mean Na+/Li+ countertransport at 150 mmol/L Na+ for the healthy individuals, when measured with the LiCl loading [mean 225 µmol Li+(hL RBC)-1], are below this cutoff value. The values obtained with the Li2CO3 method or the nystatin method are higher, although <400 µmol Li+/(hL RBC) [260 and 312 µmol Li+/(hL RBC)]. For the whole group (diabetic and nondiabetic patients) the values for V150 are significantly different when two of the three methods are compared. The V150, however, has been criticized as a marker because it was reported that at 150 mmol/L Na+ the Vmax was often not reached. Our results confirm this. Measurement of Vmax and K0.5 for Na+ could give more reliable values. Moreover, as Rutherford et al. (12) already mentioned, any differences in V150 observed can be due to differences in either Vmax or K0.5. In addition, changes in both the K0.5 and the Vmax have been reported [13, 14]. If Vmax and K0.5 are to be used as risk markers, new cutoff values for abnormal Na+/Li+ countertransport activity have to be established.
Both the Li2CO3 and nystatin loading methods result in higher values for K0.5 and Vmax than those obtained with the LiCl method (P <0.001). The reason for this is unknown. One possible explanation could be that loading with Cl- means exchange of HCO3- for Cl- via the band 3 anion transporter. This could lead to pH changes in the erythrocyte, which could influence Na+/Li+ countertransport. Elving et al., however, reported that after the washing cycles the intracellular HCO3- and Cl- concentrations were identical in cells loaded with either Li2CO3 or LiCl (6). Another possibility is that the optimal internal Li+ concentration is not reached in all experiments by this method.
Besch et al. (15) already compared the LiCl method with the Li2CO3 method. In contrast to the present study, they found no difference in values for Vmax or K0.5. But they concluded that the Li2CO3 method was to be preferred because this method takes considerably less time. Zerbini et al. (16) compared the LiCl method with the nystatin method and they concluded that at 150 mmol/L NaCl the maximum activity is not always reached, and that therefore the nystatin loading method is to be preferred. But none of these studies compared all three methods or studied the measuring error and intrasubject variation of the values obtained.
Hardman and Lant (17) and Wierzbicki (18) raised the question whether nystatin will be removed by washing. The fact that the mean values of our results obtained with both the nystatin method and Li2CO3 method do not differ indicates that the erythrocyte membranes are not damaged when nystatin is used. The same authors as well as Thomas et al. (19) questioned whether the data obtained by Zerbini et al. (16) fit to Michaelis–Menten kinetics. We found that both the data obtained with the Li2CO3 and the nystatin loading procedure do fit to Michaelis–Menten kinetics. On the other hand, the data obtained with the LiCl method showed more variation.
Rutherford et al. reported that changes in K0.5 for Na+ rather than in Vmax are the explanation for increased V150 Na+/Li+ countertransport in insulin-dependent diabetes mellitus patients with nephropathy (14). This could be due to changes in binding of Na+, but may also reflect changes in translocation rate of the transporter. But because of the large fluctuations of the K0.5 for Na+ within one subject over time (intervals of 1 month), it is doubtful whether the K0.5 can be used to identify patients at risk for development of diabetic nephropathy.
To conclude, in this study the effect of different Li+ loading methods (LiCl, Li2CO3, or nystatin) on the reproducibility of the K0.5 and Vmax values for Na+/Li+ exchange were compared. The LiCl method generates Na+/Li+ exchange activities that often do not apply to Michaelis–Menten kinetics. Our results suggest that both the nystatin method and the Li2CO3 method are preferred over the LiCl method.
Acknowledgments
We are grateful to the Dutch Diabetes Fund for supporting this work (grant 92.602).
References
The following articles in journals at HighWire Press have cited this article:
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  G. Zerbini, D. Gabellini, D. Ruggieri, and A. Maestroni Increased Sodium-Lithium Countertransport Activity: A Cellular Dysfunction Common to Essential Hypertension and Diabetic Nephropathy J. Am. Soc. Nephrol., January 1, 2004; 15(90010): S81 - 84. [Abstract] [Full Text]
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