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Na,Li-countertransport and ethnicity
AJH
1999;12:433-435
LETTERS
Na,Li-Countertransport
by Elsevier
Science, Inc.
EDITOR
and Ethnicity
The recent report by Ragone et al, which revisits the subject of ethnicity and expression of sodium-lithium countertransport (SLC) activity fails to address a number of issues.’ The use of different laboratories to determine SLC activity introduces intersite variance to interassay and intraindividual variability-which, incidentally, are not quoted. Yet there is no discussion of measures taken to counter the possible introduction of these systematic errors.’ Comparison of assay variability at the two laboratories would have been useful, although, due to the nature of the assay, may have introduced a methodologic challenge. However, as SLC activity within an individual is reasonably stable, and takes several months to respond to environmental changes,‘f3 this potential confounder could have been countered or reduced by repeated measurements of the travelling investigator’s SLC activity at both sites. The method employed by Ragone et al is a modification of that originally described by Canessa et al in 1980.4 SLC activity analysis requires the accurate determination of lithium efflux rate.2-5 Traditionally, lithium efflux rates have been determined by linear regression of repeat (duplicate or triplicate) measurements of lithium concentrations at three time points. 4-6 This approach ensures that any outlying values, which can sometimes be extreme, do not significantly affect the final estimates of efflux. .Regression analysis between two points, as used by Ragone et al, is invalid and, by using two rather than three time points, the authors assume insignificant variability, which is incorrect and may introduce substantial errors into their estimates of SLC activity.i The statistical methods employed by Ragone et al were inappropriate. SLC activity is not normally distributed within populations2,3 and yet, SLC activity data is cited as means ? SD1; median and range values are preferred.2,3 Similarly, triglycerides and insulin concentrations are not normally distributed, hence direct analysis by ANOVA is inappropriate. Nonparametric testing or log transformation of data followed by analysis of the derived curves for skew and kurtosis prior to ANOVA analysis are required. Furthermore, despite differences in gender distribution across study groups, no adjustment was made for this variable in the ANOVA analysis although adjustment was made for age. Gender is known to have a significant impact on SLC activity, in contrast to age, which does not appear to be an important determinant.3,7
Published
TO THE
Ragone et al found an association between SLC activity, high blood pressure, and metabolic abnormalities in Italian subjects but only with hypertension in the urbanized Chinese subjects.’ However, the Chinese cohort is subject to strikingly different environmental influences, including dietary fat and salt intakes. Without a comparison with properly matched groups of subjects using a 2 X 2 matrix study design, it is impossible to say whether elevated countertransport activity would also have been seen in Chinese subjects with similar metabolic (abnormalities. Failure to incorporate these methodologic controls within their study may explain why their results, which suggest increased SLC activity in Chinese subjects compared with Italian subjects, differs from those obtained during our own investigation into the impact of ethnicity on SLC in subjects sharing similar environments.8 In 1985, an investigation into the expression of SLC activity in hypertensive, hyperlipidemic, and hypertensive hyperlipidemic subjects concluded that dyslipidemia and hypertension are independently associated with elevations in countertransport activity.’ This observation has not been fully explained; however, the answer may lie within the characterization of the countertransporter. It is generally accepted that SLC activity provides only limited information on the operation of the countertransporter. Kinetic characterization of the countertransporter is now recognized as being superior to determination of activity alone. The two kinetic measures that serve to determine SLC activity, maximal rate of turnover (V,,,) and kNa, appear associated with plasma lipid concentrations and hypertension, respectively. Ragone et al found consistent increases in activity and attributed this to an increase in V,,, being associated with metabolic abnormalities, such as dyslipidemia or insulin resistance, whilst the increase in activity in hypertensive patients may be associated with a reduced k,,. Kinetic approaches have been applied to the study of ethnic differences in SLC activity.8 These showed no difference in either countertransport activity, V,,,, or k,, between Chinese or European subjects recruited within the UK. This finding implies that the elevation observed by Ragone et al reflects the influence of different environmental, as opposed to genetic, factors on their two study cohorts. Kinetic analysis may have differentiated more clearly between ethnic, environ-
0895-7061/99/$20.00
434
LETTERS
TO
THE
AlH-APRIL
EDITOR
mental, and disease related influences in these two populations. In conclusion, the study of Ragone et al used dated methods with an insufficiently robust study design.’ Conclusions from this study on the roles of ethnicity, environment, or disease in determining SLC activity can but be severely restricted by inclusion of these limitations. REFERENCES 1.
Ragone E, Strazzullo P, Siani A, et al: Ethnic differences in red blood cell sodium/lithium countertransport and metabolic correlates of hypertension: an international collaborative study. Am J Hypertens 1998;11:935-941.
2.
Rutherford PA, Thomas TH, Wilkinson R: Erythrocyte sodium-lithium countertransport: clinically useful, pathophysiologically instructive or just phenomenology? Clin Sci 1992;82:341-352.
3.
Hardman TC, Lant AF: erythrocyte sodium-lithium tens 1996;14:695-703.
4.
Canessa M, Adragna N, Solomon HS, et al: Increased sodium-lithium countertransport in red cells of patients with essential hypertension. N Engl J Med 1980;302:772776.
5.
Na,Li-Countertransport
of lim-
and Ethnicity:
We thank you very much for giving us the opportunity to reply to the observations by Drs. Hardman and Wierzbicki concerning our paper on the ethnic differences in red blood cell Na/Li countertransport (CT) in hypertension.’ Actually, most of these observations are quite surprising, eg, the complaint about the lack of measures aiming to reduce possible intersite variance in the comparison of Chinese and Italian measures of Na/Li CT. First of all, contrary to Hardman and Wierzbicki’s statement, we did quote in our paper the interassay and intraassay variability of Na/Li CT as being always
12, NO.
4, PART
1
itations and assumptions of traditional and kinetic methodologies. J Memb Biol 1998;161:197-205. Canessa M: Kinetic properties of Na+/H’ exchange and Li’/Na’, Na+/Na+, and Na+/Li’ exchanges of human red cells. Methods Enzymol 1989;173:176-191. Hardman TC, Dubrey SW, Soni S, Lant AF: Relation of sodium-lithium countertransport activity to markers of cardiovascular risk in normotensive subjects. J Hum Hypertens 1995;9:589-596. Hardman TC, Croft I’, Morrish Z, et al: Kinetic characteristics of the erythrocyte sodium-lithium countertransporter in black normotensive subjects compared with three other ethnic groups. J Hum Hypertens 1998;12:2934. Corrocher R, Steinmayr M, Ruzzenente 0, et al: Elevation of red cell sodium-lithium countertransport in hyperlipidemias. Life Sci 1985;36:649-655.
Controversies surrounding countertransport. J Hyper-
Hardman TC, Thomas T, Lant AF: Characterization the erythrocyte sodium-lithium countertransporter:
1999%VOL.
From
the Department Imperial
T.C. HARDMAN AND A. WIERZBICKI of Cardiovascular Medicine, College School of Medicine, Charing Cross Hospital, London, England.
Address correspondence and reprint requests to T.C. Hardman, Department of Cardiovascular Medicine, Imperial College School of Medicine, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, England. 0 1999 American Journal of Hypertension, Ltd. PI1 SO895-7061(99)00034-5
Reply Another technical question raised by Hardman and Wierzbicki concerns our use of only two (0 and 60 min) rather than three time points for the determination of the activity of Na/Li CT. Although in principle one should acknowledge, as we do, that the efflux rates determined by linear regression of three measurements can afford greater accuracy, the question in epidemiologic studies is to choose the least expensive (in terms of time and cost of reagents and personnel) procedure reliable enough for the purpose of the study. In repeated tests conducted in our laboratory, the advantage of making three (duplicate) rather than two (duplicate) measurements of Li-efflux rate was negligible. The same line of reasoning applies to the decision as to whether or not to perform a kinetic characterization of the countertransporter. There is no doubt that, generally speaking, the determination of the maximal rate of turnover (V,,,) together with the affinity constant for sodium may be more informative than the simple measurement of the Li-efflux rate. This, however, is not easily applicable to population studies. We do not believe that the method we used, ie, the measurement of the Li-efflux rate, is dated. It is just a compromise, and a good compromise if one remembers that this was the method used in longitu-
1999;12:433-435
LETTERS
Na,Li-Countertransport
by Elsevier
Science, Inc.
EDITOR
and Ethnicity
The recent report by Ragone et al, which revisits the subject of ethnicity and expression of sodium-lithium countertransport (SLC) activity fails to address a number of issues.’ The use of different laboratories to determine SLC activity introduces intersite variance to interassay and intraindividual variability-which, incidentally, are not quoted. Yet there is no discussion of measures taken to counter the possible introduction of these systematic errors.’ Comparison of assay variability at the two laboratories would have been useful, although, due to the nature of the assay, may have introduced a methodologic challenge. However, as SLC activity within an individual is reasonably stable, and takes several months to respond to environmental changes,‘f3 this potential confounder could have been countered or reduced by repeated measurements of the travelling investigator’s SLC activity at both sites. The method employed by Ragone et al is a modification of that originally described by Canessa et al in 1980.4 SLC activity analysis requires the accurate determination of lithium efflux rate.2-5 Traditionally, lithium efflux rates have been determined by linear regression of repeat (duplicate or triplicate) measurements of lithium concentrations at three time points. 4-6 This approach ensures that any outlying values, which can sometimes be extreme, do not significantly affect the final estimates of efflux. .Regression analysis between two points, as used by Ragone et al, is invalid and, by using two rather than three time points, the authors assume insignificant variability, which is incorrect and may introduce substantial errors into their estimates of SLC activity.i The statistical methods employed by Ragone et al were inappropriate. SLC activity is not normally distributed within populations2,3 and yet, SLC activity data is cited as means ? SD1; median and range values are preferred.2,3 Similarly, triglycerides and insulin concentrations are not normally distributed, hence direct analysis by ANOVA is inappropriate. Nonparametric testing or log transformation of data followed by analysis of the derived curves for skew and kurtosis prior to ANOVA analysis are required. Furthermore, despite differences in gender distribution across study groups, no adjustment was made for this variable in the ANOVA analysis although adjustment was made for age. Gender is known to have a significant impact on SLC activity, in contrast to age, which does not appear to be an important determinant.3,7
Published
TO THE
Ragone et al found an association between SLC activity, high blood pressure, and metabolic abnormalities in Italian subjects but only with hypertension in the urbanized Chinese subjects.’ However, the Chinese cohort is subject to strikingly different environmental influences, including dietary fat and salt intakes. Without a comparison with properly matched groups of subjects using a 2 X 2 matrix study design, it is impossible to say whether elevated countertransport activity would also have been seen in Chinese subjects with similar metabolic (abnormalities. Failure to incorporate these methodologic controls within their study may explain why their results, which suggest increased SLC activity in Chinese subjects compared with Italian subjects, differs from those obtained during our own investigation into the impact of ethnicity on SLC in subjects sharing similar environments.8 In 1985, an investigation into the expression of SLC activity in hypertensive, hyperlipidemic, and hypertensive hyperlipidemic subjects concluded that dyslipidemia and hypertension are independently associated with elevations in countertransport activity.’ This observation has not been fully explained; however, the answer may lie within the characterization of the countertransporter. It is generally accepted that SLC activity provides only limited information on the operation of the countertransporter. Kinetic characterization of the countertransporter is now recognized as being superior to determination of activity alone. The two kinetic measures that serve to determine SLC activity, maximal rate of turnover (V,,,) and kNa, appear associated with plasma lipid concentrations and hypertension, respectively. Ragone et al found consistent increases in activity and attributed this to an increase in V,,, being associated with metabolic abnormalities, such as dyslipidemia or insulin resistance, whilst the increase in activity in hypertensive patients may be associated with a reduced k,,. Kinetic approaches have been applied to the study of ethnic differences in SLC activity.8 These showed no difference in either countertransport activity, V,,,, or k,, between Chinese or European subjects recruited within the UK. This finding implies that the elevation observed by Ragone et al reflects the influence of different environmental, as opposed to genetic, factors on their two study cohorts. Kinetic analysis may have differentiated more clearly between ethnic, environ-
0895-7061/99/$20.00
434
LETTERS
TO
THE
AlH-APRIL
EDITOR
mental, and disease related influences in these two populations. In conclusion, the study of Ragone et al used dated methods with an insufficiently robust study design.’ Conclusions from this study on the roles of ethnicity, environment, or disease in determining SLC activity can but be severely restricted by inclusion of these limitations. REFERENCES 1.
Ragone E, Strazzullo P, Siani A, et al: Ethnic differences in red blood cell sodium/lithium countertransport and metabolic correlates of hypertension: an international collaborative study. Am J Hypertens 1998;11:935-941.
2.
Rutherford PA, Thomas TH, Wilkinson R: Erythrocyte sodium-lithium countertransport: clinically useful, pathophysiologically instructive or just phenomenology? Clin Sci 1992;82:341-352.
3.
Hardman TC, Lant AF: erythrocyte sodium-lithium tens 1996;14:695-703.
4.
Canessa M, Adragna N, Solomon HS, et al: Increased sodium-lithium countertransport in red cells of patients with essential hypertension. N Engl J Med 1980;302:772776.
5.
Na,Li-Countertransport
of lim-
and Ethnicity:
We thank you very much for giving us the opportunity to reply to the observations by Drs. Hardman and Wierzbicki concerning our paper on the ethnic differences in red blood cell Na/Li countertransport (CT) in hypertension.’ Actually, most of these observations are quite surprising, eg, the complaint about the lack of measures aiming to reduce possible intersite variance in the comparison of Chinese and Italian measures of Na/Li CT. First of all, contrary to Hardman and Wierzbicki’s statement, we did quote in our paper the interassay and intraassay variability of Na/Li CT as being always
12, NO.
4, PART
1
itations and assumptions of traditional and kinetic methodologies. J Memb Biol 1998;161:197-205. Canessa M: Kinetic properties of Na+/H’ exchange and Li’/Na’, Na+/Na+, and Na+/Li’ exchanges of human red cells. Methods Enzymol 1989;173:176-191. Hardman TC, Dubrey SW, Soni S, Lant AF: Relation of sodium-lithium countertransport activity to markers of cardiovascular risk in normotensive subjects. J Hum Hypertens 1995;9:589-596. Hardman TC, Croft I’, Morrish Z, et al: Kinetic characteristics of the erythrocyte sodium-lithium countertransporter in black normotensive subjects compared with three other ethnic groups. J Hum Hypertens 1998;12:2934. Corrocher R, Steinmayr M, Ruzzenente 0, et al: Elevation of red cell sodium-lithium countertransport in hyperlipidemias. Life Sci 1985;36:649-655.
Controversies surrounding countertransport. J Hyper-
Hardman TC, Thomas T, Lant AF: Characterization the erythrocyte sodium-lithium countertransporter:
1999%VOL.
From
the Department Imperial
T.C. HARDMAN AND A. WIERZBICKI of Cardiovascular Medicine, College School of Medicine, Charing Cross Hospital, London, England.
Address correspondence and reprint requests to T.C. Hardman, Department of Cardiovascular Medicine, Imperial College School of Medicine, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, England. 0 1999 American Journal of Hypertension, Ltd. PI1 SO895-7061(99)00034-5
Reply Another technical question raised by Hardman and Wierzbicki concerns our use of only two (0 and 60 min) rather than three time points for the determination of the activity of Na/Li CT. Although in principle one should acknowledge, as we do, that the efflux rates determined by linear regression of three measurements can afford greater accuracy, the question in epidemiologic studies is to choose the least expensive (in terms of time and cost of reagents and personnel) procedure reliable enough for the purpose of the study. In repeated tests conducted in our laboratory, the advantage of making three (duplicate) rather than two (duplicate) measurements of Li-efflux rate was negligible. The same line of reasoning applies to the decision as to whether or not to perform a kinetic characterization of the countertransporter. There is no doubt that, generally speaking, the determination of the maximal rate of turnover (V,,,) together with the affinity constant for sodium may be more informative than the simple measurement of the Li-efflux rate. This, however, is not easily applicable to population studies. We do not believe that the method we used, ie, the measurement of the Li-efflux rate, is dated. It is just a compromise, and a good compromise if one remembers that this was the method used in longitu-