Vascular and Neurological Changes in Early Diabetes: Advances in Metabolic Disorders, Vol. 2

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I

Macroangiopathy

Macroangiopathy — Its Prevalence in Asymptomatic Diabetes

H. Keen*† and R.J. Jarrett*,     *Department of Medicine, Guy’s Hospital Medical School, London, England; †Invited speaker

Publisher Summary

This chapter describes the prevalence of macroangiopathy in asymptomatic diabetes. Disease of large and medium-sized arteries has come to be accepted as a major consequence of diabetes mellitus, occurring particularly in middle aged and older patients, and accounting for much of the morbidity and mortality of maturity-onset diabetics. The chapter describes a study in which a newly discovered diabetic group was characterized by 2 h blood sugars of 200 mg/100 ml or more. A normal control group was constituted of individuals with 2 h blood sugar levels below 120 mg/100 ml. Among men, there was no clear trend in the blood sugar category in individual age/sex groups. Among women, all certified cardiovascular deaths occurred either in diabetic or borderline subjects, but only in one age group did the numbers support the view that this was other than a chance association. In terms of cardiovascular mortality, there was no significant difference between the normal and the borderline subjects.

Disease of large and medium-sized arteries has come to be accepted as a major consequence of diabetes mellitus, occurring particularly in middle-aged and older patients and accounting for much of the morbidity and mortality of maturity-onset diabetics. This relationship does not go unchallenged, however, and it is disturbing that such astute observers as Mitchell and Schwartz (1965) and Pyke (1968) remain unconvinced of the reality of the association. Nor does the relationship occur universally. In Japan (Kuzuya and Kosaka, 1971; Goto and Fukuhara, 1968) and in other low atherosclerosis prevalence groups (Shaper et al., 1962), the incidence of coronary and peripheral arterial disease is also low among diabetics—little if at all higher than among corresponding nondiabetic population groups, and lower than among even the nondiabetic peoples of Europe and North America. Despite this clear-cut geographical difference in macrovascular disease, the frequency of microvascular disease is at least as high in Asian diabetic populations as in comparable Western groups.

Whatever its explanation, this dissociation of macro- and microvascular disease in Japanese and Western diabetics seriously weakens the argument that assigns an important role to the latter in the pathogenesis of the former. It also weakens the argument of those who ascribe impaired glucose tolerance to the effects of arterial disease, rather than vice versa. The dissociation supports the hope that atherosclerosis is not an inevitable consequence of diabetes in man, an observation that can be set alongside the assertion of Schettler (1970) of the absence of coronary disease in hypotensive diabetics. This apparent difference in atherosclerotic disease between East and West provides an important opportunity for carefully controlled transnational comparisons and a challenge, already perceived by Japanese colleagues, to try to examine the extent and the means by which Westernization of their way of life permeates the arterial wall as it penetrates the structure of the society.

In Europe, North America, and other Westernized populations, however, the increased prevalence of atherosclerosis in diabetics appears established beyond reasonable doubt. The relationship is most clear and can be partially quantified in epidemiologically based studies such as ours in Bedford, England (Keen et al., 1965), and those in Tecumseh, Michigan (Ostrander et al., 1965), Framingham, Massachusetts, (Kannel et al., 1967), Paris, France (Gelin et al., 1967), and Busselton, Australia (Welborn et al., 1968). These prevalence studies of atherosclerosis as it affects the coronary, limb, and cerebral circulation all point to an approximately 2 to 2l/2-fold excess involvement in the diabetic.

Prevalence studies tell only part of the story, however. They give a selective picture which emphasizes the lesser manifestations, underestimating those rendering the subject unavailable for study. A much clearer picture of the relationship is given by incidence studies which collect all the manifestations of disease over a period of time. There are very few published studies on the incidence of arterial disease in diabetic populations compared with suitable nondiabetic control groups. The main substance of this communication is to give the preliminary results of a 5-year incidence study in the three population groups defined in 1962 following the Bedford survey. The three glycemia groups were defined solely on the basis of their blood sugar levels measured 2 hours after 50 gm oral glucose on capillary blood using the AutoAnalyzer microferricyanide analytical method.

Methods and Materials

The three groups were defined as follows. A newly discovered diabetic group was characterized by 2-hour blood sugars of 200 mg/100 ml or more. A normal control group was constituted of individuals with 2-hour blood sugar levels below 120 mg/100 ml. It matched, by age and sex, an intermediate or borderline diabetic group with 2-hour blood sugars between 120 and 199 mg/100 ml. The newly found diabetics were referred to their family doctors after detection in 1962, and they determined further management with or without the help of the local diabetes clinic. These diabetics were re called for systematic review in 1967, and information on defaulters was sought out by a variety of means. The normal control subjects were similarly recalled approximately 12–15 months later and subjected to similar examination and also an investigation of defaulters.

The borderline group has been, and remains, under continuous scrutiny with regular visits to follow-up clinics every 6 months since 1962. For the purposes of this study, only the records of their visits in the first half of 1967 were referred to in order to make the degree of ascertainment comparable to that in the other two groups. Comparisons were based upon the application of standard WHO questionnaires regarding cardiovascular events (Rose and Blackburn, 1968)—angina pectoris, myocardial infarction, and intermittent claudication—to members of all three glycemia groups, and upon the certified cause of death of those who died during the 5-year follow-up period. Mortality ascertainment for the three groups was a little short of 100%. For morbidity information, however, it was 95% for the borderline group, 88% for the diabetics, and 82% for the normal controls.

Results

Figure 1 summarizes the 1962 prevalence estimates of arterial disease in the three glycemia groups, aggregated after suitable corrections for differences in age and sex composition. A similarly age/sex-adjusted aggregate of clinical arterial incidents, lethal and nonlethal, occurring during the follow-up period is shown in Fig. 2. This latter comparison is composed of questionnaire positive manifestations of morbidity and of deaths certified as cardiovascular in cause over the follow-up period, but does not include ECG changes. The gradient of arterial incidents with blood sugar is highly significant statistically (p < 0.01) and is steeper than the slope for prevalence. When the incidence aggregates are broken down into the component elements of morbidity and mortality by age and sex, however, the trend becomes less striking, although other features emerge.

FIG. 1 Percentage frequency of arterial disease prevalence in representative unselected newly found diabetics, borderline diabetics, and normoglycemic age/scx-matched controls defined in the 1962 Bedford survey. Allowance has been made for small age and sex differences between the groups. Arterial disease estimates are based upon numbers of individuals with questionnaire evidence of arterial symptoms and/or Minnesota codable ECG changes indicating coronary disease. The trend of prevalence with glycemia category is statistically significant (p < 0.05). The continuous line represents, in diagrammatic form, the positively skewed normal distribution of glucose tolerance/intolerance in the general population.

FIG. 2 Percentage frequency of individuals in the three glycemia groups sustaining one or more arterial events during the 5 years 1962–1967. Arterial events include the new appearance of questionnaire positive symptoms and/or death certified as cardiovascular in cause. As in Fig. 1, allowance has been made for age and sex differences between groups. The trend is statistically significant (p < 0.01). The continuous line represents, in diagrammatic form, the positively skewed normal distribution of glucose tolerance/intolerance in the general population.

Percentage mortality certified as due to cardiovascular cause in the three glycemia groups by age and sex is shown in Table I. Among men there is no clear trend in the blood sugar category in individual age/sex groups. Among women all certified cardiovascular deaths occurred either in diabetic or borderline subjects, but only in one age group did the numbers support the view that this was other than a chance association. In terms of cardiovascular mortality, there was no significant difference between the normals and the borderline subjects.

TABLE I

PERCENTAGE MORTALITY BY SEX, AGE GROUP AND BLOOD SUGAR CATEGORY CERTIFIED As DUE TO CARDIOVASCULAR CAUSE DURING THE 5-YEAR PERIOD 1962–1967 FOLLOWING THE BEDFORD SURVEY

aSignificant excess in diabetics (p < 0.05).

Similar comparisons by age and sex in respect to cardiovascular morbidity are shown in Table II. Here there is a much clearer trend of arterial abnormality with blood sugar group, particularly among women. In two of the age groups, this trend achieves statistical significance. The striking aggregate trend shown in Fig. 2 thus owes a great deal to the gradient of morbidity and, to a lesser extent, of mortality in women.

TABLE II

PERCENTAGE BY SEX, AGE GROUP AND BLOOD SUGAR CATEGORY RESPONDING POSITIVELY WITH QUESTIONNAIRE EVIDENCE OF A NEW NONLETHAL ARTERIAL EVENT DURING THE 1962–1967 PERIOD FOLLOWING THE BEDFORD SURVEY

aTrends significant (p < 0.05).

Discussion

Our findings resemble in some respects those of Epstein (1967), who reported on the incidence of fatal and nonfatal coronary disease after an average follow-up period of 4 years from the Tecumseh survey. His figures show that for men there was no trend in coronary heart disease incidence with the 1-hour blood sugar measured at the initial survey. For women, however, the trend was very apparent across the whole age range. Additional data come from the Framingham study in which Kannel and colleagues (1967) reported on the incidence of coronary heart disease in men and women aged 30–59 years who developed diabetes during the 12 years of follow-up from the first survey time point. Coronary disease showed greater incidence, in terms of both morbidity and mortality, in diabetics compared with nondiabetics. Its special impact in women was clearly apparent. A clear excess of coronary death in men was indicated by the almost threefold increase in diabetics compared with controls in the 10-year prospective study of 370 diabetics employed by the DuPont company (Pell and D’Alonzo, 1970).

Conclusion

Data from Japan suggest that there atherosclerotic disease affects the diabetic much less commonly than in the West and is not an integral part of the diabetic syndrome.

In contrast with Japan, where atherosclerotic disease prevalence and incidence is low in general (Kimura and Keys, 1970) and little if at all increased in the diabetic, in Western countries (with their much higher general prevalence and incidence) the diabetic appears to be two or three times more likely to be affected than the nondiabetic. This special liability to arterial disease is also seen in people with impaired glucose tolerance in general, and there appears to be some relationship between the degree of impairment and the heightening of the risk. Women are especially vulnerable because of loss of the relative immunity that nonhyperglycemic women enjoy.

The geographical dissociation of diabetes mellitus and atherosclerotic disease suggests that other factors—genetic or environmental—must operate before the atheroma proneness of the diabetic becomes manifest. A progressive Westward trend of Japanese figures suggests that environmental influences (e.g., nutritional factors) rank high on the suspect list.

ACKNOWLEDGMENTS

The Bedford study is supported by a grant from the Department of Health and Social Security. We are grateful to many helpers, in both Bedford and London, and for the willing cooperation of the subjects of the study.

REFERENCES

Epstein, F.H. Proc. Roy. Soc. Med. 1967; 60:56.

Gelin, J., Elgrishi, I., Ducimetiere, P., Richard, J.L. Bull. Inst. Nat. Sante Rech. Med. 1967;22(No. 2).

Goto, Y., Fukuhara, N. J. Jap. Diabetes Soc. 1968; 11:197.

Kannel, W.B., Castelli, W.P., McNamara, P.M. J. Occup. Med. 1967; 9:611.

Keen, H., Rose, G.A., Pyke, D.A., Boyns, D.R., Chlouverakis, C., Mistry, S. Lancet. 1965; 2:505.

Kimura, N., Keys, A. Circulation. 1970;41/42(Suppl. 1):1–101 (Amer. Heart Ass. Monogr. No. 29.)

Kuzuya, N., Kosaka, K. Tsuji S., Wada M., eds. Diabetes Mellitus in Asia, 1970. Excerpta Med. Found.: Amsterdam, 1971:11.

Mitchell, J.R.A., Schwartz, C.J. Arterial Disease,. Oxford: Blackwell, 1965; 308.

Ostrander, L.D., Jr., Francis, T., Jr., Hayner, N.S., Kjelsberg, M.O., Epstein, F.H. Ann. Intern. Med. 1965; 62:1188.

Pell, S., D’Alonzo, C.A. J. Amer. Med. Ass. 1970; 214:1833.

Pyke, D.A. Oakley W.G., Pyke D.A., Taylor K.W., eds. Clinical Diabetes and its Biochemical Basis. Blackwell: Oxford, 1968; 530.

Rose, G.A., Blackburn, H. World Health Organ. Monogr. Ser. (No. 56):1968.

Schettler, G. (1970). In Atherosclerosis, Proc. 2nd Int. Symp. (R. J. Jones, ed.). Springer-Verlag, Berlin and New York.

Shaper, A.G., Lee, K.T., Scott, R.F., Goodale, F., Thomas, W.A. Amer. J. Cardiol. 1962; 10:390.

Welborn, T.A., Curnow, D.H., Wearne, J.T., Cullen, K.J., McCall, M.G., Stenhouse, N.S. Med. J. Aust. 1968; 2:778.

The Prevalence of Asymptomatic Diabetes in Human Atherosclerosis

Kenneth J. Kingsbury*,     *Invited speaker; St. Mary’s Hospital, London, England

Publisher Summary

This chapter focuses on the prevalence of asymptomatic diabetes in human atherosclerosis. In an experiment described in the chapter, aortoiliac and femoropopliteal atherosclerosis were routinely studied by pulse changes and arteriograms in contrast to coronary or cerebral atherosclerosis. Pulse changes often did not distinguish between the different kinds of obstruction or the extent of the narrowing disease. It was necessary to see if a relationship existed between reduced glucose tolerances and aortoiliac and femoropopliteal atherosclerosis as seen on arteriograms. The irregularities were classified as extensive if confluent irregularities existed throughout an artery, slight if the arteries were smooth walled or had an isolated patch of irregularity less than 1 ½ inches in length, and moderate for intermediate degrees. Similar relationships were obtained in pilot studies in which blood glucose concentrations were compared to the measured lengths of irregularity summed for the aortoiliac and femoropopliteal regions or the percentage of aortoiliac and femoropopliteal arterial walls that could be seen to be irregular.

The prevalence of reduced glucose tolerances in middle-aged populations and the frequency of symptoms of ischemic vascular disease have been studied by several groups, as summarized previously (Kingsbury, 1968).

Our studies have been concerned with whether a relationship exists between reduced tolerances and atherosclerosis, whether this differs with the type and extent of the arterial lesions, and whether this can explain the apparently varying prevalence noted by Wahlberg (1966).

Our investigations were therefore restricted to patients in whom at least some estimate could be obtained of the type and extent of the arterial lesions that underlaid the symptoms.

In contrast to coronary or cerebral atherosclerosis, aortoiliac and femoropopliteal atherosclerosis were routinely studied by pulse changes and arteriograms. However, pulse changes often did not distinguish between the different kinds of obstruction or the extent of the narrowing disease. Therefore it was necessary to see if a relationship existed between reduced glucose tolerances and aortoiliac and femoropopliteal atherosclerosis as seen on arteriograms. The criteria for the selection of patients have been described elsewhere (Kingsbury, 1971).

A series of studies showed first that atherosclerosis—as seen on arteriograms—could be classified into irregularities of the arterial wall, occlusions, and stenoses. Their proportions allowed each type to be subdivided into extensive and slight forms, with an intermediate grade interposed for tests of linearity. The severe and the minimal grades can never be confused and, as the intermediate grade is intermediate in all age, biochemical, blood group, and prognostic parameters examined so far, it appeared possible that the grades can be accepted as simple three-point scales of the disease (Kingsbury, 1969).

The number of occlusions or stenoses in the aortoiliac and femoropopliteal arteries were simply counted on the arteriograms, checked by a general examination of the pulses, and graded as 0, 1, 2, or more.

The irregularities were classified as extensive if confluent irregularities existed throughout an artery, slight if the arteries were smooth-walled or had an isolated patch of irregularity less than 1½ inches in length, and moderate for intermediate degrees. These differences are shown in Fig. 1.

FIG. 1 Arteriograms showing (a) slight, (b) moderate, and (c) extensive atheromatous irregularities of the aortoiliac arteries.

It is interesting that the percentage of patients with severe irregularities but patent arteries (10%), or multiple occlusions with minimal atheroma (8%), corresponded to the percentages obtained at postmortem for similar disease states in the coronary arteries of people who have died from acute coronary ischemia (Crawford et al., 1961).

The results to be shown were derived from this classification, but similar relationships were obtained in pilot studies in which blood glucose concentrations were compared to the measured lengths of irregularity summed for the aortoiliac and femoropopliteal regions, or the percentage of aortoiliac and femoropopliteal arterial walls that could be seen to be irregular (Kingsbury, 1966a).

Our experience (Kingsbury, 1968) and the study of the Birmingham College of General Practitioners (1963) showed that prevalence rates were affected not only by the level selected as a margin between normal and abnormal but also by the time after the glucose load at which samples were taken. In a middle-aged population, the glucose concentration in blood samples taken 1 hour or less after 50 gm of glucose by mouth was affected more by age and less by atherosclerosis than samples taken after 2 hours (Kingsbury, 1968).

It appeared that the early and late aspects of the glucose tolerance curve were subject to separate mechanisms, differently affected by age and atherosclerosis, and that the 2-hour level was more suitable for studies of the disease.

A 2-hour glucose level of 110–115 mg/100 ml was best found to distinguish individuals with slight and extensive atherosclerotic irregularities (from a total of over 600 patients). This was very close to the conventionally accepted value of 120 mg/100 ml which can therefore be adopted because of the advantage of comparisons with other studies.

The comparisons between the prevalence of asymptomatic diabetes, age, and the type and extent of atherosclerosis were therefore based on arterio-graphic classifications, blood samples taken 2 hours after 50 gm of glucose by mouth, and a blood glucose concentration of 120 mg/100 ml accepted as the upper limit of normal.

The incidence of blood glucose concentrations over 120 mg/100 ml was found to be much higher in patients with extensive atheromatous irregularities than in those with only minimal (slight) disease (Kingsbury, 1966b) (Fig. 2). This was not simply the result of a nonspecific worsening of the whole biochemistry as the disease progressed. Plasma cholesterol levels did not change markedly with the degree of atherosclerosis. In addition, blood sugar concentrations did not change significantly with the number of occlusions.

FIG. 2 The blood glucose concentrations, plotted against age, 2 hours after 50 gm of glucose by mouth in patients with slight, moderate, and extensive arterial irregularities.

This selective relationship was checked in a further study (Kingsbury et al., 1969) as shown in Fig. 3. It can be seen that once again blood glucose concentrations rose markedly with the degree of irregularity but showed little change with the number of occlusions. In contrast, plasma cholesterol levels showed no marked changes with either type of disease.

FIG. 3 The 2-hour blood glucose concentration in patients with slight, moderate, and extensive arterial irregularities, and 0, 1, and 2+ occlusions plotted against age.

The relationship between blood glucose concentration and atherosclerotic irregularities was obvious when the percentages of abnormal biochemical values in each disease group were compared: Blood glucose <120 mg/100 ml, plasma cholesterol >275 mg/100 ml, plasma cholesteryl linoleic acid <35% (Fig. 4).

FIG. 4 The percentage of patients with abnormal 2-hour blood glucose concentrations (> 120 mg/100 ml), plasma cholesterol concentrations (> 275 mg/100 ml), and plasma cholesteryl linoleic acid concentrations (< 35%) in patients with slight. moderate, or extensive irregularities, and 0, 1, and 2+ occlusions or stenoses.

It can be seen that, although the prevalence of asymptomatic diabetes was unaffected by the number of stenoses or occlusions, it increased markedly with the degree of mural atheroma. This is in contrast to the percentage of abnormally high plasma cholesterol values and the percentage of abnormally low plasma cholesteryl linoleic concentrations. The blood glucose irregularity relationship was highly significant in all studies (p < 0.001), whether tested by the usual chi-square tests, or by covariance analysis in which the overall variations of glucose tests were considered (Kingsbury, 1968).

However, as shown in both the Birmingham (1963) and Bedford (Keen, 1964) studies, the prevalence of high 2-hour values rose with age—although less than the 1-hour concentrations. This also applied to our patients over 60-65 years old (Kingsbury, 1968).

In all our studies the proportion of patients with extensive irregularities (to which blood glucose concentrations are related) also rose sharply in subjects over 60-65 years of age. The question arose therefore whether the greater prevalence of elevated 2-hour blood sugar values in the older age groups was simply the result of increasing age, or whether it was linked more with the development of mural atheroma.

The prevalence of high 2-hour glucose values in three age categories within each disease group is shown in Fig. 5. The percentages of abnormally high plasma cholesterol levels are included for comparison. [The reasons for the selection of these particular age groups have been discussed previously (Kingsbury, 1968).]

FIG. 5 The proportions of patients with slight, moderate, or extensive irregularities in various age groups from three different studies.

It can be seen that the prevalence of abnormally high blood glucose concentrations did not rise at all with age in patients with only slight atheroma. However, in those with severe irregularities (in contrast to the high plasma cholesterol values), the prevalence of asymptomatic diabetes rose sharply to over 80% in the oldest group.

It appears therefore that the prevalence of asymptomatic diabetes, associated with claudication, was linked specifically with the development of mural irregularities in the aortoiliac and femoropopliteal arteries. This also seems to apply to the incidence of acute coronary ischemia.

The percentages of patients with slight, moderate, and extensive atheromatous irregularities (and 0, 1, and 2+ occlusions in the aortoiliac and femoropopliteal arteries) who developed myocardial infarction in the first 3 years of their follow-up are shown in Fig. 6. Myocardial infarction was defined by the typical clinical history and by significant Q-wave alterations (Kingsbury et al., 1969). In both disease types (mural irregularity and lumenal occlusions), the incidence of myocardial infarction was three to four times higher in those with extensive disease than in those with only slight atheroma or patent arteries. At each disease level, however, the incidence of infarction was similar in patients with abnormally high or normal blood glucose concentrations. No independent relationship appeared between the blood sugar level and the incidence of infarction in these middle-aged patients once the amount of their arterial disease was considered. Similar results were indicated in a previous study (Kingsbury, 1966a).

FIG. 6 The percentage of patients with slight, moderate, or extensive arterial irregularities and 0, 1, and 2+ occlusions who developed myocardial infarction, separately for those with normal and abnormal blood glucose, plasma cholesterol, and plasma cholesteryl linoleic acid concentrations.

These results suggest that the blood sugar levels per se had little effect on the development of ischemic events but reflected some functional disturbance associated only with the type of atherosclerosis seen on arteriograms as irregularities of the arterial wall.

This helps to explain the varying prevalence of asymptomatic diabetes in middle age and in patients presenting with coronary, aortoiliac, or femoropopliteal disease. It depends (at least in part) not on the symptoms, age, or even on the disease as a whole, but on the extent of the underlying, often hidden, mural atheroma.

Although this raises new and difficult requirements for the assessment of both normal subjects and patients, it does appear to provide a specific relationship whereby the prevalence of asymptomatic diabetes, and its nature, causes, and treatments, can be investigated more precisely.

REFERENCES

Birmingham College of General Practitioners. Brit. Med. J. 1963; ii:655.

Crawford, T., Dexter, D., Teare, R.D. Lancet. 1961; i:181. [450].

Keen, H. Proc. Roy. Soc. Med. 1964; 57:200.

Kingsbury, K.J. J. Ther. 1966; 1:50.

Kngsbury, K.J. Lancet. 1966; ii:1374.

Kingsbury, K.J. Post Grad. Med. J. 1968; 44(Suppl. 1):944.

Kingsbury, K.J. Nature. 1969; 224:146.

Kingsbury, K.J. Lancet. 1971; i:199.

Kingsbury, K.J., Morgan, D.M., Stovold, R., Brett, C.G., Anderson, J. Lancet. 1969; ii:1325.

Wahlberg, F. Acta Med. Scand. Suppl. 1966; 455:180.

A Search for Macroangiopathy in Patients with Latent Diabetes*

Stefan S. Fajans†‡, Peter N. Weissman†, Park W. Willis, III§, John C. Floyd, Jr.†, Sumer Pek† and J.W. Conn†,     †Department of Internal Medicine (Division of Endocrinology and Metabolism and the Metabolism Research Unit), The University of Michigan, Ann Arbor, Michigan; ‡Invited speaker; §Department of Internal Medicine (Division of Cardiology), The University of Michigan, Ann Arbor, Michigan

Publisher Summary

This chapter discusses macroangiopathy in patients with latent diabetes. In an experiment described in the chapter, the results of the initial diagnostic glucose tolerance tests performed on patients aged 9–17 at diagnosis were shown. The results of the highest glucose tolerance tests obtained in these subjects were presented. Plasma levels of insulin for 15 of these patients who were nonobese were also presented. After administration of glucose, the diabetic group exhibited a significantly subnormal increase in plasma insulin. The mean sum of increments in plasma insulin above fasting levels for all six intervals of the test was significantly less than for control subjects. Results of the glucose tolerance tests performed on patients aged 18–25 years at diagnosis were shown. The mean increase in plasma insulin in 21 of these patients who were nonobese was delayed. 17 of these 21 patients had subnormal levels similar to those of younger patients, while 4 showed responses which, although delayed in reaching peak levels, were greater than the mean response of control