- Email: [email protected]
Sodium intake and mortality
CORRESPONDENCE
correlating reported salt intake with measured output is shown in a study in which a select group of educated and motivated healthy volunteers on an ad lib diet were asked to keep contemporaneous dietary diaries while also collecting daily 24-h urine samples.2 On one occasion, average daily urinary sodium excretion increased by over 50%, but the source of the increased sodium intake could not be found in the food diaries made after each meal. The same quartile also reported exceptionally low calorie intake even though they were quite overweight (body-mass index >25 kg/m2). These questionable data, which form the basis for Alderman’s article, might be explained if they were very poor estimators of their diet, or if, rather than their actual intake, they reported what they thought they should have been eating, given their much higher hypertension and heart disease rates. Powerful computers and sophisticated statistical software packages enable investigators to do massive number-crunching with ease, but the recipe for this type of research should be flavoured with a tincture of commonsense. In the end, Alderman and colleagues are correct when they stated that their study “does not justify any particular recommendations”. Karl Engelman 20 Turnberry Lane, Hilton Head Island, SC 29928, USA 1
2
Alderman MH, Cohen H, Madhavan S. Dietary sodium intake and mortality: the National Health and Nutritional Examination Survey (NHANES I). Lancet 1998; 351: 781–85. Mattes RD, Christensen CM, Engelman K. Effects of hydrochlorothiazide and amiloride on salt taste and excretion (intake). Am J Hypertens 1990; 3: 436–43.
Sir—Rather than shed new light on sodium intake and mortality, Michael Alderman and colleagues’ report1 brings unnecessary confusion into the discussion on the relation between dietary sodium and mortality. Alderman and co-workers used a single 24 hour dietary recall on nutrient intake in 1971–75 to estimate an individual’s long-term intake of sodium and calories. Their findings show that the investigators have failed in their attempt to find out whether dietary sodium is associated with mortality in a general population. They argue that middle-aged people are likely to have a stable nutrient intake over many years. If so, how do they explain the fact that most people in their lowest sodium quartile survived the 20 year follow-up period at a calorie intake that should have resulted in death from starvation?
THE LANCET • Vol 351 • May 16, 1998
Among women in the lowest sodium quartile, the calculated calorie intake was only 989 kcal, which is about 50% of the recommended daily allowance of 1800–2100 kcal for women in this age group. Among men in the lowest sodium quartile, the calculated calorie intake was 1473 kcal, which is 50–60% of the recommended dietary daily allowance of 2300–2800 kcal for men in this age group. For the maintenance of basal metabolism only, middle-aged women require at least 1200 kcal daily, and middle-aged men at least 1500 kcal per day. Any physical activity increases the calorie requirement. We were surprised that the women on the low calorie intake were, on the average, 4 kg (6%) heavier than those in the highest sodium quartile, despite a two-fold calorie intake in the latter group. Hence, the calculations and conclusions have been based on invalid data. The investigators overlook evidence that the dramatic decrease in all-cause and cardiovascular mortality among the 5 million population of Finland during the past 20 years reflects the decrease in sodium intake, and some increase in the intake of potassium.2-4 Since the 1970s, changes in the intake of sodium have been carefully monitored by measurement of 24 hour urinary sodium excretion in representative population samples. As a result of the continuous decrease, the average intake of sodium is now about 30% lower than it was 20 years ago. Although there has been an increase in obesity, consumption of alcohol and smoking among women between 1972 and 1992, there has been a 10 mm Hg fall in the population average of diastolic blood pressure, and about 60% decrease in deaths from stroke and ischaemic heart disease among men and women aged 30–59 years.2-4 Only 0·5–0·6 mm Hg of the fall of the population blood pressure could be attributed to antihypertensive drug treatment.4 The decrease in sodium intake and in the dietary sodium-topotassium ratio in Finland is the main factor accounting for the 9·5 mm Hg fall in the population average of diastolic blood pressure. Evidence that a general reduction of dietary sodium in the population is beneficial is based on hard mortality endpoints. During the same period that the average intake of sodium has decreased 30%, the life expectancy of Finns has increased by about 5 years. *Heikki Karppanen, Eero Mervaala Institute of Biomedicine, Department of Pharmacology and Toxicology, University of Helsinki, FIN-00014, Helsinki, Finland
1
2
3
4
Alderman MH, Cohen H, Madhavan S. Dietary sodium intake and mortality: the National Health and Nutrition Examination Survey (NHANES 1). Lancet 1998; 351: 781–85. Vartiainen E, Puska P, Pekkanen J, Tuomilehto J, Jousilahti P. Changes in risk factors explain changes in mortality from stroke in Finland. BMJ 1994; 309: 23–27. Vartiainen E, Sarti C, Tuomilehto J, Kuulasmaa K. Do changes in cardiovascular risk factors explain changes in mortality from stroke in Finland? BMJ 1995; 310: 901–04. Karppanen H, Mervaala E. Adherence to and population impact of nonpharmacological and pharmacological antihypertensive therapy. J Hum Hypertens 1996; 10(suppl 1): S57–S61.
Authors’ reply Sir—Hugh de Wardener and Graham MacGregor ignore the significant inverse association of sodium to allcause mortality (p=0·0069) reported from NHANES 1 and note the borderline significance (p=0·086) of sodium to cardiovascular (CVD) mortality. The values we reported were for the full Cox model. In the best-fit model, in which variables with a p value greater than 0·10 were eliminated, sodium showed a highly significant inverse association with CVD mortality: the hazard ratio was 0·86 (95% CI 0·81–0·93); p<0·0001. Although common practice is to report the best-fit model, we conservatively chose to present the full model. The independent associations, in opposite directions, of sodium and sodium/calorie to mortality, probably reflect different physiological events. On the basis of additional analyses, we can now report that the inverse sodium to mortality associations persisted within each sodium/calorie quartile. de Wardener and MacGregor also note the imprecision with which table salt is estimated in NHANES 1. In multivariate analysis, the responses (always, sometimes, never) were not significantly associated with mortality, which probably reflects the small contribution of table salt compared with salt consumed within food. For discretionary salt to change the results of our study, it would be necessary for those reporting low table salt to have eaten a high sodium diet, which contradicts reports that individuals who have low-sodium diets also tend to use less table salt and vice versa.1 de Wardener and MacGregor repeat a previous, inaccurate, statement2 about baseline sodium in our study of hypertensives,3 which we have already answered at length.4 Briefly, patients were uniformly advised to maintain
1509
CORRESPONDENCE
usual diet while avoiding high-sodium foods. Their sodium intake matched that seen in US Intersalt sites. Inaccurate assessments were likely to have been distributed throughout the population, resulting in an underestimation of the true magnitude of the observed inverse relation of sodium to events. Karl Engelman comments on the unreliable estimation of sodium intake by dietary recall. We agree that NHANES 1 probably underestimated sodium. The real concern, however, is not that across-the-board under reporting occurred, but whether errors in reporting of sodium biased the observed relation. We used stratified and multivariate analyses to account for the possibility that sicker patients might have consumed less sodium and died sooner. The inverse relation of sodium to mortality held. We are now able to report two further analyses. Exclusion of 2161 participants who reported recent change in appetite or dietary alteration produced sodium to mortality results for the restricted group consistent with the whole. When we excluded 1926 participants who reported less than a 1000 kcal intake daily, the results were similar. Heikki Karppanen and Eero Mervaala refer to ecological reports of communities with aggregate changes in several behaviours that have enjoyed a coincidental decline in mortality. Sodium intake was not measured in the studies cited, but referenced from different, serial cross-sectional studies. There were concurrent changes in dietary fat and other nutrients. Without direct linkage of various changes in the characteristics of individuals to their outcomes, it is mere speculation to attribute a decline in mortality to one of the many factors (sodium) that changed. *Michael H Alderman, Hillel Cohen, Shantha Madhavan Epidemiology and Social Medicine, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
Impaired clearance of oxidised proteins in neurodegenerative diseases Sir—Marilynn Larkin’s Feb 14 feature (p 424)1 on protein abnormalities in some neurodegenerative diseases raises the possibility that mutant proteins entering the nucleus are not removed by the overworked cell. Accumulation of abnormal proteins may be a feature of all neurodegenerative disorders, certainly so for Alzheimer’s and prion diseases as well as those discussed by Larkin. Raised concentrations of protein carbonyls, a marker of oxidative damage to proteins by various mechanisms, are observed in all neurodegenerative diseases studied to date,2,3 and are frequently taken as evidence of increased free radical formation in these disorders. However, increases in oxidised proteins could equally well result from failure to clear them, a process normally undertaken by the proteasome complex.4 Indeed, certain products generated by freeradical attack on lipids in the presence of proteins can block proteasome action. 5 In Parkinson’s disease, concentrations of protein carboynls are raised in all brain areas,3 suggesting that abnormalities of proteolysis can spread beyond areas of primary pathology. We postulate that defects in clearance of proteins, either because they resist proteolysis (eg, amyloid peptides and abnormal prion proteins) or because proteolytic systems are defective, inhibited or overloaded, is a common feature of all neurodegenerative diseases. There may, of course, be regional variations in the susceptibility of neurons to injury by accumulation of abnormal proteins. For example, raised concentrations of protein carbonyls are seen throughout the brain in Parkinson’s disease,3 but pathological change is largely restricted to the substantia nigra.
(e-mail: [email protected])
*Barry Halliwell, Peter Jenner
1
Neurodegenerative Disease Research Centre, King’s College London, London SW3 6LX, UK
2
3
4
McCarron DA, Morris CD, Stanton JL. Hypertension and calcium. Science 1984; 226: 386–89. MacGregor GA. Low urinary sodium and myocardial infarction. Hypertension 1996; 27: 156. Alderman MH, Madhavan S, Cohen H, Sealey JE, Laragh JH. Low urinary sodium is associated with greater risk of myocardial infarction among treated hypertensive men. Hypertension 1995; 25: 1144–52. Alderman MH, Laragh JH. Low urinary sodium and myocardial infarction. Hypertension 1996; 27: 156–57.
1510
1 2
3
4
Larkin M. Therapeutic target reveals itself in diseased neurons. Lancet 1998; 351: 424. Smith CD, Carney JM, Starke-Reed PE, et al. Excess brain protein oxidation and enzyme dysfunction in normal aging and Alzheimer’s disease. Proc Natl Acad Sci USA 1997; 88: 10540–43. Alam ZI, Daniel SE, Lees AJ, Marsden DC, Jenner P, Halliwell B. Generalized increase in protein carbonyls in the brain in Parkinson’s but not incidental Lewy body disease. J Neurochem 1997; 69: 1326–29. Grune T, Davies KJA. Breakdown of
5
oxidized proteins as a part of secondary antioxidant defences in mammalian cells. Biofactors 1997; 6: 165–72. Conconi M, Friguet B. Proteasome inactivation upon ageing and on oxidation— effect of HSP 90. Mol Biol Rep 1997; 24: 45–50.
Refutation of investigation commissioned by Karolinska Institute Sir—The refutation by Ulf Lönn (Jan 31, p 375)1 about alleged scientific misconduct could suggest some ambiguity about the role of the Karolinska Institute and its scientific expert committee with respect to the fact that Lönn’s published results are scientifically invalid and should be retracted. We wish to clarify the situation. Lönn now claims a 73% success rate of his PCR-based analysis for gene amplification in fixed fine-needle aspirates and formalin-fixed specimens from bladder cancer. This rate is contradicted by tables IV–VI in ref 2, in which all of 555 analyses are reported as successful. The PCR technique consistently used only 20 cycles of amplification of 1 g total nucleic acid in raw-extracts. But the electrophoretic bands were far too strong to correspond to the low amount of hardly detectable gene material theoretically expected from Lönn’s procedure. Lönn delivered photographic negatives of his electrophoresis of alleged amplified genes, mainly c-ERB-B2. These were all cut from unknown sources, lacked identification, and thus provided no proof that the analyses of specified patients samples had been done. Contrary to his statement not one intact photograph of an identifiable successful electrophoresis had been saved by Lönn, who has admitted, but not published, that after 1993 no PCR analyses were successful. Patients with advanced breast cancer with or without gene amplification had 15-year survivals of about 15% and 95%, respectively—a remarkable difference, which virtually disappeared when causes of death were taken from the official cancer registry rather than by Lönn’s own assessment. Lönn’s statement is true that his technicians remembered attempts to do immunohistochemistry of c-ERB-B2 protein, but it is also true that they could not confirm that they were successful. No protocols were kept and all relevant slides have disappeared. At least 4000 densitometric measurements should have been done in the 4 years when Lönn explored gene amplifications as a prognostic marker. The expert committee estimated that
THE LANCET • Vol 351 • May 16, 1998
correlating reported salt intake with measured output is shown in a study in which a select group of educated and motivated healthy volunteers on an ad lib diet were asked to keep contemporaneous dietary diaries while also collecting daily 24-h urine samples.2 On one occasion, average daily urinary sodium excretion increased by over 50%, but the source of the increased sodium intake could not be found in the food diaries made after each meal. The same quartile also reported exceptionally low calorie intake even though they were quite overweight (body-mass index >25 kg/m2). These questionable data, which form the basis for Alderman’s article, might be explained if they were very poor estimators of their diet, or if, rather than their actual intake, they reported what they thought they should have been eating, given their much higher hypertension and heart disease rates. Powerful computers and sophisticated statistical software packages enable investigators to do massive number-crunching with ease, but the recipe for this type of research should be flavoured with a tincture of commonsense. In the end, Alderman and colleagues are correct when they stated that their study “does not justify any particular recommendations”. Karl Engelman 20 Turnberry Lane, Hilton Head Island, SC 29928, USA 1
2
Alderman MH, Cohen H, Madhavan S. Dietary sodium intake and mortality: the National Health and Nutritional Examination Survey (NHANES I). Lancet 1998; 351: 781–85. Mattes RD, Christensen CM, Engelman K. Effects of hydrochlorothiazide and amiloride on salt taste and excretion (intake). Am J Hypertens 1990; 3: 436–43.
Sir—Rather than shed new light on sodium intake and mortality, Michael Alderman and colleagues’ report1 brings unnecessary confusion into the discussion on the relation between dietary sodium and mortality. Alderman and co-workers used a single 24 hour dietary recall on nutrient intake in 1971–75 to estimate an individual’s long-term intake of sodium and calories. Their findings show that the investigators have failed in their attempt to find out whether dietary sodium is associated with mortality in a general population. They argue that middle-aged people are likely to have a stable nutrient intake over many years. If so, how do they explain the fact that most people in their lowest sodium quartile survived the 20 year follow-up period at a calorie intake that should have resulted in death from starvation?
THE LANCET • Vol 351 • May 16, 1998
Among women in the lowest sodium quartile, the calculated calorie intake was only 989 kcal, which is about 50% of the recommended daily allowance of 1800–2100 kcal for women in this age group. Among men in the lowest sodium quartile, the calculated calorie intake was 1473 kcal, which is 50–60% of the recommended dietary daily allowance of 2300–2800 kcal for men in this age group. For the maintenance of basal metabolism only, middle-aged women require at least 1200 kcal daily, and middle-aged men at least 1500 kcal per day. Any physical activity increases the calorie requirement. We were surprised that the women on the low calorie intake were, on the average, 4 kg (6%) heavier than those in the highest sodium quartile, despite a two-fold calorie intake in the latter group. Hence, the calculations and conclusions have been based on invalid data. The investigators overlook evidence that the dramatic decrease in all-cause and cardiovascular mortality among the 5 million population of Finland during the past 20 years reflects the decrease in sodium intake, and some increase in the intake of potassium.2-4 Since the 1970s, changes in the intake of sodium have been carefully monitored by measurement of 24 hour urinary sodium excretion in representative population samples. As a result of the continuous decrease, the average intake of sodium is now about 30% lower than it was 20 years ago. Although there has been an increase in obesity, consumption of alcohol and smoking among women between 1972 and 1992, there has been a 10 mm Hg fall in the population average of diastolic blood pressure, and about 60% decrease in deaths from stroke and ischaemic heart disease among men and women aged 30–59 years.2-4 Only 0·5–0·6 mm Hg of the fall of the population blood pressure could be attributed to antihypertensive drug treatment.4 The decrease in sodium intake and in the dietary sodium-topotassium ratio in Finland is the main factor accounting for the 9·5 mm Hg fall in the population average of diastolic blood pressure. Evidence that a general reduction of dietary sodium in the population is beneficial is based on hard mortality endpoints. During the same period that the average intake of sodium has decreased 30%, the life expectancy of Finns has increased by about 5 years. *Heikki Karppanen, Eero Mervaala Institute of Biomedicine, Department of Pharmacology and Toxicology, University of Helsinki, FIN-00014, Helsinki, Finland
1
2
3
4
Alderman MH, Cohen H, Madhavan S. Dietary sodium intake and mortality: the National Health and Nutrition Examination Survey (NHANES 1). Lancet 1998; 351: 781–85. Vartiainen E, Puska P, Pekkanen J, Tuomilehto J, Jousilahti P. Changes in risk factors explain changes in mortality from stroke in Finland. BMJ 1994; 309: 23–27. Vartiainen E, Sarti C, Tuomilehto J, Kuulasmaa K. Do changes in cardiovascular risk factors explain changes in mortality from stroke in Finland? BMJ 1995; 310: 901–04. Karppanen H, Mervaala E. Adherence to and population impact of nonpharmacological and pharmacological antihypertensive therapy. J Hum Hypertens 1996; 10(suppl 1): S57–S61.
Authors’ reply Sir—Hugh de Wardener and Graham MacGregor ignore the significant inverse association of sodium to allcause mortality (p=0·0069) reported from NHANES 1 and note the borderline significance (p=0·086) of sodium to cardiovascular (CVD) mortality. The values we reported were for the full Cox model. In the best-fit model, in which variables with a p value greater than 0·10 were eliminated, sodium showed a highly significant inverse association with CVD mortality: the hazard ratio was 0·86 (95% CI 0·81–0·93); p<0·0001. Although common practice is to report the best-fit model, we conservatively chose to present the full model. The independent associations, in opposite directions, of sodium and sodium/calorie to mortality, probably reflect different physiological events. On the basis of additional analyses, we can now report that the inverse sodium to mortality associations persisted within each sodium/calorie quartile. de Wardener and MacGregor also note the imprecision with which table salt is estimated in NHANES 1. In multivariate analysis, the responses (always, sometimes, never) were not significantly associated with mortality, which probably reflects the small contribution of table salt compared with salt consumed within food. For discretionary salt to change the results of our study, it would be necessary for those reporting low table salt to have eaten a high sodium diet, which contradicts reports that individuals who have low-sodium diets also tend to use less table salt and vice versa.1 de Wardener and MacGregor repeat a previous, inaccurate, statement2 about baseline sodium in our study of hypertensives,3 which we have already answered at length.4 Briefly, patients were uniformly advised to maintain
1509
CORRESPONDENCE
usual diet while avoiding high-sodium foods. Their sodium intake matched that seen in US Intersalt sites. Inaccurate assessments were likely to have been distributed throughout the population, resulting in an underestimation of the true magnitude of the observed inverse relation of sodium to events. Karl Engelman comments on the unreliable estimation of sodium intake by dietary recall. We agree that NHANES 1 probably underestimated sodium. The real concern, however, is not that across-the-board under reporting occurred, but whether errors in reporting of sodium biased the observed relation. We used stratified and multivariate analyses to account for the possibility that sicker patients might have consumed less sodium and died sooner. The inverse relation of sodium to mortality held. We are now able to report two further analyses. Exclusion of 2161 participants who reported recent change in appetite or dietary alteration produced sodium to mortality results for the restricted group consistent with the whole. When we excluded 1926 participants who reported less than a 1000 kcal intake daily, the results were similar. Heikki Karppanen and Eero Mervaala refer to ecological reports of communities with aggregate changes in several behaviours that have enjoyed a coincidental decline in mortality. Sodium intake was not measured in the studies cited, but referenced from different, serial cross-sectional studies. There were concurrent changes in dietary fat and other nutrients. Without direct linkage of various changes in the characteristics of individuals to their outcomes, it is mere speculation to attribute a decline in mortality to one of the many factors (sodium) that changed. *Michael H Alderman, Hillel Cohen, Shantha Madhavan Epidemiology and Social Medicine, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
Impaired clearance of oxidised proteins in neurodegenerative diseases Sir—Marilynn Larkin’s Feb 14 feature (p 424)1 on protein abnormalities in some neurodegenerative diseases raises the possibility that mutant proteins entering the nucleus are not removed by the overworked cell. Accumulation of abnormal proteins may be a feature of all neurodegenerative disorders, certainly so for Alzheimer’s and prion diseases as well as those discussed by Larkin. Raised concentrations of protein carbonyls, a marker of oxidative damage to proteins by various mechanisms, are observed in all neurodegenerative diseases studied to date,2,3 and are frequently taken as evidence of increased free radical formation in these disorders. However, increases in oxidised proteins could equally well result from failure to clear them, a process normally undertaken by the proteasome complex.4 Indeed, certain products generated by freeradical attack on lipids in the presence of proteins can block proteasome action. 5 In Parkinson’s disease, concentrations of protein carboynls are raised in all brain areas,3 suggesting that abnormalities of proteolysis can spread beyond areas of primary pathology. We postulate that defects in clearance of proteins, either because they resist proteolysis (eg, amyloid peptides and abnormal prion proteins) or because proteolytic systems are defective, inhibited or overloaded, is a common feature of all neurodegenerative diseases. There may, of course, be regional variations in the susceptibility of neurons to injury by accumulation of abnormal proteins. For example, raised concentrations of protein carbonyls are seen throughout the brain in Parkinson’s disease,3 but pathological change is largely restricted to the substantia nigra.
(e-mail: [email protected])
*Barry Halliwell, Peter Jenner
1
Neurodegenerative Disease Research Centre, King’s College London, London SW3 6LX, UK
2
3
4
McCarron DA, Morris CD, Stanton JL. Hypertension and calcium. Science 1984; 226: 386–89. MacGregor GA. Low urinary sodium and myocardial infarction. Hypertension 1996; 27: 156. Alderman MH, Madhavan S, Cohen H, Sealey JE, Laragh JH. Low urinary sodium is associated with greater risk of myocardial infarction among treated hypertensive men. Hypertension 1995; 25: 1144–52. Alderman MH, Laragh JH. Low urinary sodium and myocardial infarction. Hypertension 1996; 27: 156–57.
1510
1 2
3
4
Larkin M. Therapeutic target reveals itself in diseased neurons. Lancet 1998; 351: 424. Smith CD, Carney JM, Starke-Reed PE, et al. Excess brain protein oxidation and enzyme dysfunction in normal aging and Alzheimer’s disease. Proc Natl Acad Sci USA 1997; 88: 10540–43. Alam ZI, Daniel SE, Lees AJ, Marsden DC, Jenner P, Halliwell B. Generalized increase in protein carbonyls in the brain in Parkinson’s but not incidental Lewy body disease. J Neurochem 1997; 69: 1326–29. Grune T, Davies KJA. Breakdown of
5
oxidized proteins as a part of secondary antioxidant defences in mammalian cells. Biofactors 1997; 6: 165–72. Conconi M, Friguet B. Proteasome inactivation upon ageing and on oxidation— effect of HSP 90. Mol Biol Rep 1997; 24: 45–50.
Refutation of investigation commissioned by Karolinska Institute Sir—The refutation by Ulf Lönn (Jan 31, p 375)1 about alleged scientific misconduct could suggest some ambiguity about the role of the Karolinska Institute and its scientific expert committee with respect to the fact that Lönn’s published results are scientifically invalid and should be retracted. We wish to clarify the situation. Lönn now claims a 73% success rate of his PCR-based analysis for gene amplification in fixed fine-needle aspirates and formalin-fixed specimens from bladder cancer. This rate is contradicted by tables IV–VI in ref 2, in which all of 555 analyses are reported as successful. The PCR technique consistently used only 20 cycles of amplification of 1 g total nucleic acid in raw-extracts. But the electrophoretic bands were far too strong to correspond to the low amount of hardly detectable gene material theoretically expected from Lönn’s procedure. Lönn delivered photographic negatives of his electrophoresis of alleged amplified genes, mainly c-ERB-B2. These were all cut from unknown sources, lacked identification, and thus provided no proof that the analyses of specified patients samples had been done. Contrary to his statement not one intact photograph of an identifiable successful electrophoresis had been saved by Lönn, who has admitted, but not published, that after 1993 no PCR analyses were successful. Patients with advanced breast cancer with or without gene amplification had 15-year survivals of about 15% and 95%, respectively—a remarkable difference, which virtually disappeared when causes of death were taken from the official cancer registry rather than by Lönn’s own assessment. Lönn’s statement is true that his technicians remembered attempts to do immunohistochemistry of c-ERB-B2 protein, but it is also true that they could not confirm that they were successful. No protocols were kept and all relevant slides have disappeared. At least 4000 densitometric measurements should have been done in the 4 years when Lönn explored gene amplifications as a prognostic marker. The expert committee estimated that
THE LANCET • Vol 351 • May 16, 1998