Studies on normal blood glucose level—Statistical approach to interpretation of glucose tolerance test

J Chron Dis 1976, Vol. 29, pp. 129-140. Pergamon Press. Printed in Great Britain

STUDIES ON NORMAL BLOOD GLUCOSE LEVELSTATTSTICAL APPROACH TO INTERPRETATION OF GLUCOSE TOLERANCE TEST AKIRA SASAKIand NARUTO HORIUCHI Center for Adult Diseases, Osaka, Japan (Received

in revised,form

14 April

1975)

INTRODUCTION INTERPRETATION of the glucose tolerance test (GTT) has long been a matter of controversial opinion without reaching general agreement. Mosenthal and Barry [l], Moyer and Womack [2] and Unger et al. [3] attempted to establish a normal range of the blood glucose level, on the basis of observations of its distribution in GTT for apparently healthy subjects. Their attempt was followed by Wilkerson [4, 51 and Fajans and Conn [6, 71 with few modifications, resulting in the advent of diagnostic criteria of diabetes mellitus which have been very widely employed today. As has been repeatedly pointed out, clinical features of diabetes mellitus in Japan are quite different from those in Western countries, because of the differences in race, environmental factors and dietary habits [8-lo]. Therefore, it has also been indicated that it is questionable if the diagnostic criteria thus determined in the Western countries are applicable to Japanese people without any modification. This question also comes up from epidemiological observations on relations among the mortality, morbidity, and clinical course of diabetics in Japan. Presently, it was attempted to establish new criteria which are primarily meant for the Japanese population. In the diagnostic criteria so far employed, including those proposed by the committee on diagnostic criteria of the Japan Diabetic Society [l 11,it has been considered very important to set a severe diagnostic level so that overlooking of very mild cases may be prevented. However, generally speaking, in setting diagnostic criteria, a special emphasis should be laid on the following points: (1) The diagnostic level is to be determined on the basis of a statistical analysis of distribution of the blood glucose level. (2) The diagnostic criteria thus determined are to be confirmed by a long-term follow-up study. In addition, since glucose tolerance is obviously related to aging, age should be taken into account as a factor in establishing the criteria.

129

130

AKIRA SASAKI and NARUTO HORIUCHI

AND

MATERIAL

METHOD

Those who were studied were 586 apparently healthy males admitted for a health examination to the Center for Adult Diseases, Osaka, excluding those with obvious abnormal findings. Their age distribution is given in Table 1. Clinical courses were observed of 272 subjects including mild diabetics, who were admitted for health check-up 5-7 yr previously. They were recalled for re-examination, similar to those given at their initial hospitalization. TABLE

I.

AGE DISTRIBUTION OF SUBJECTS Age

Number

30-39

103

40-49

238

50-59

178

60-69

60

70-

7

Total

586

In the glucose tolerance test, 50 g of glucose were given orally and venous blood specimens were collected from the cubital vein. Glucose was measured in whole blood by Autoanalyzer (Hoffman’s method). RESULTS 1.

Separating the normal from the abnormal

In general, the frequency distribution of the measurements of many clinical examinations may be classified schematically into three types as given in Fig. 1. The first type shows two independent distributions of normal and abnormal groups giving a clear cut separation (Fig. l(a)). The second type is a bimodal distribution, indicating an overlap between the two groups in various degrees. By assuming two curves, we provide an optimum separation point so that the probability of misclassification could be minimized as given in Fig. l(b). The third type is a unimodal but asymmetric distribution where no information would be obtained to separate the abnormal from the normal. In this case it has been usual to draw an arbitrary line for separation. However, as the committee on statistics of the American Diabetes Association suggested [12], it is assumed that a separation might be obtained if a normal group had a symmetrical distribution. Namely, as given in Fig. I(c), the left ascending part of the frequency distribution could be used in extending the right descending part of the hypothetical distribution curve of the ‘normal’ group. The remaining part of the original distribution would represent the abnormal group, and hypothetical distribution could be obtained by replotting from the baseline. The distribution of the blood glucose level usually belongs to the third type and, in fact, the data obtained in this study also indicated a unimodal distribution skewed to the right side. Therefore, it is reasonable to adopt this idea in separating the normal from the abnormal.

Studies on Normal Blood Glucose Level

s

I

Two independent

1

B~modal

131

dtstrlbutions

dlstrlbutlon Diagnostic

range

Abnormal

/ False Unlmodai

\

negative

‘False posltlve

skewed dlstrlbutlon

Measurement

FIG. 1. Mode of distribution

of measurement

for normal and abnormal populations.

2. Estimation of a normal range of blood glucose

In applying the above-stated idea to the present data, some modification was made. The cumulative per cent distribution of the blood glucose level, rather than the original frequency distribution, was plotted on a normal probability paper. A straight line was eye-fitted to the part up to 50% of the cumulative distribution, which corresponds to the left ascending part of the original distribution, as given in Fig. 2. This method gives not only a theoretically equivalent result to that described above, but proves to be more practical in handling data. Furthermore, it has an advantage that the estimated mean (a 50% point) and the standard deviation (a 15.8% point) can be obtained directly from the graph, on the basis of the property of the normal probability paper. For the estimation of a normal range, however, a range of two standard deviations around the mean was adopted, regardless of the distribution of the abnormal. This method was considered also more practical than the original one in which the upper limit was largely dependent on the distribution of the abnormal as seen in Fig. l(c). This procedure was performed for each age group as to fasting, l-, 2- and 3-hr levels. As an example, procedures of the 2-hr level for an age group 40-49 yr was shown in Fig. 3. The mean, the standard deviation and the upper limit of a normal range (mean +2 S.D.) of the blood glucose level are summarized in Table 2. The upper limit of fasting and three-hour levels thus obtained was 90-100 mg/lOO ml,

AKIRASASAKI and NARUTO HORIUCHI

132

Blood

FIG. 2. Estimating

procedure

glucose

of the parameters

level,

on the normal deviation.

mg/lOOml

probability

paper,

8

: mean, S : standard

with a slight increase with aging, showing a good agreement with formerly accepted criteria. However, it should be noticed that l- and 2-hr levels for groups with over 40 yr of age were obviously higher than formerly thought to be the case, indicating 180-190 mg/ 100 ml and 140-150 mg/lOO ml, respectively, with an increase as age advanced. The present values, following a 50 g glucose load, were higher by 20 mg and 30 mg/lOO m for the I- and 2-hr values respectively than the comparable values reported by Wilkerson [4] after a 100 g glucose load. This is all the more surprising in view of the fact that the Autoanalyzer-Hoffman method gives slightly higher values than the SomogyiNelson method [l 11. Similarly, those were higher by 30 mg/lOO ml both in l- and 2-hr levels than in the Fajans-Conn criteria [251 with a loading of 1.75 g of glucose per kilogram of ideal body weight. Moreover, these two values are markedly higher than in the criteria recommended by WHO [14] and by the committee on diagnostic criteria of the Japan Diabetic Society [l l] (Table 3). As a matter of fact, such a pattern of GTT indicating a relatively high level in a l- and 2-hr value is found very frequently in multiphasic health examination or epidemiological study [15]. 3. Follow-up study of bloodglucose

level

Follow-up glucose tolerance tests were performed 7 yr later in the subjects. Figure 4 compares the distribution of fasting and 2-hr blood sugars in the two studies. The initial level was classified as indicated on the vertical axis of the figure, and the present level was scaled on the abscissa. The shadowed area denotes subjects whose present

Studies on Normal Blood Glucose Level TABLE 2. MEAN, STANDARD

133

DEVIATION AND UPPER-LIMIT OF BLEND GLUCOSE LEVEL BY AGE GROUP h3W

ml)

Year

Fasting

30-39

40-49

50-59

60-69

11.2

76.5

18.2

81.5

7.0

9.0

9.5

10.2

91.2

94.5

97.2

101.9

113.5

118.0

118.0

129.0

30.0

31.5

33.5

30.0

173.5

181.0

185.0

189.0

Mean

82.4

84.0

90.0

98.0

SD.

20.0

27.5

27.0

28.0

122.5

139.0

144.0

154.0 72.0

Mean S.D. Upper limit

I-hr

Mean SD. Upper limit

2-hr

Upper limit 3-hr

Mean

69.0

70.5

69.5

S.D.

10.5

9.5

13.0

14.0

Upper limit

90.0

89.5

95.5

100.0

*Upper limit=Mean+2

60

S.D.

.’

Ll -

70 60 50 40

/

/'

F;=8&0 S=275

x+25=139-0

Two-hour

FIG. 3. Estimation

blood

glucose

level,

mg/lOO ml

of the mean and the standard deviation from the cumulative frequency distribution of the blood glucose level (2-hr level, in the age group 4Ck-49yr); 8: mean, S: standard deviation.

AURA SASAKI and NARUTOHORIUCHI

134

level was higher than the initial one, the blank area subjects whose present level was lower than the initial one. As given in Fig. 4(a) in fasting, those who indicated previously a low blood glucose level tended to show a high level presently, resulting in an increase of the shadowed area, and vice versa. It was noteworthy that the mode of distribution was found to be around 90 mg/lOO ml regardless of the initial level. This type of variation is known as the ‘regression phenomenon’ and implies that the initial higher level was not abnormal.

TABLE 3. DIAGNOSTIC CRITERIA RECOMMENDED ‘ov~wmA~mE~'~~0~0seD

Normal value* (mg/lOOml)

BY THE JAPAN DIABETIC SOCIETY AND CRITERIA FOR BYTHE AUTHORS(~EETEXT)

Diabetic value* (mg/lOOml)

Overt diabetic value (mg/lOOml)

Fasting

100

1 hr

140

160

220

2 hr 3 hr

100

130

160 120

120

*Values recommended by Japan Diabetic Society; 50g glucose load and Somogyi-Nelson method on whole blood.

l-•-

:::.\ 2 3 m 6E D

2012ONl39 o-. iO14ONl59

8 0

o-=’

P t :: LA_

16Om

2O o

Fasting the

FIG. 4(a). The distribution

follow-up

blood glucose examination,

level

at

mg/fOO

ml

of blood glucose level of the follow-up examination blood glucose level at the initial examination,

as grouped by the

135

Studies on Normal Blood Glucose Level

i

I

n =25 .-A-

60

70

, 90

90

100

, , aA._ 110 Ix) I30

Two-hour blood glucose the follow-up examlnatlon, I-k.

4(b). The distribution

I40

150

level ot mg/lCOml

of blood glucose level of the follow-up examination blood glucose level at the initial examination.

as grouped by the

In the 1-hr level and the 2-hr level, the variability was also observed in the same fashion as in the fasting level though the present distributions were somewhat dispersed (Fig. 4(b)). The 3-hr level resulted in the same way as the fasting level. These phenomena, which have been often reported [16-191 as an individual variation or sometimes a spontaneous remission of an abnormal carbohydrate metabolism, implies that the blood glucose level varies within a relatively wide range, where the presently estimated upper limits of the normal blood glucose level are included. 4. Prediction of the risk of developing diabetes On the other hand, as seen in Fig. 4, it was also found that some of those who had a higher blood glucose level at the initial test developed diabetic conditions obviously showing a high level at the follow-up test. Of the 272 subjects whose clinical courses during follow-up period were examined, those who exceeded the value given in Table 3 (third column) were assumed to have developed diabetes. These values were arbitrarily decided, but assumed to be acceptable without argument as values for ‘overt diabetes’. The rates of deterioration were given in Table 4 in relation to the initial blood glucose level for four points of GTT separately. The rates of subsequent development of ‘overt diabetes’ were less than 3’;,;, for those with 110 mg/llO ml or less in fasting at the initial test, but the rates increased markedly when the value of the initial test exceeded this level. A similar trend was observed in 1,2 and 3 hr, where the critical values for subsequent deterioration proved to be 160, 120 and 110 mg/lOO ml respectively. In other words, for those exceeding

136

AKIRA SA~AKIand NARUTO HORIUCHI TABLE 4.

RATE OF DETERIORATION BY INITIALBLOODGLUCOSELEVEL (a) Fasting

Blood glucose level (mg/dl) - 79 80-

89

90-

99

lW109

No. exam.

No. developed

%

33 76 72

2

2.78

46

1

2.17

110-119

25

3

12.00

120-

20

8

40.00

(b) One-hour level -119

47

1

3.23

120-139

46

1

2.17

140-159

66

3

4.55

160-179

49

7

14.29

180-199

28

4

14.29

200-219

10

4

40.00

220-

13

10

76.92

(c) Two-hour level - 79

18

80- 99

42

100-l 19

92

3

5.44

120-139

62

13

20.97

140-159

29

5

17.24

160-

28

14

50.00

(d) Three-hour

level

- 69

36

IO- 79

50

1

SO- 89

52

1

1.92

90-

46

2

4.34

99

2.00

100-109

34

2

5.88

110-119

17

2

11.76

120-

16

10

62.50

the above values, a possible development of diabetes would be expected in a probability of 12-20%, suggesting a possibility of using these values for ‘predictive diagnostic criteria’. At any rate, these values happened to be very close to those recommended by the committee for diagnostic criteria of the Japan Diabetic Society. DISCUSSION

It is generally accepted nowadays that the onset of diabetes is predetermined hereditarily, and before the onset there is a long latent period of the disease, detection of which is beyond the capability of today’s examination techniques [6, 7, 21, 223. This

Studies on Normal Blood Glucose Level

137

period can be only retrospectively confirmed when the patient actually reveals clinical manifestations, never prospectively. Therefore, the only practical way is to examine their blood glucose level in an attempt to find minimal abnormalities even in an epidemiological study, where detection of potential cases is most desirable. But it was found that if the so far adopted criteria were uniformly applied, a considerable number of nondiabetic cases would be included in a diabetic group. Since the urgent need was to rule out such nondiabetic cases, the present authors’ aim was directed to the establishment of practical diagnostic criteria for an epidemiological study or health examination. As mentioned before, the blood glucose values given in Table 3 show appreciably higher values than previously considered. These values were obtained of normal subjects and were confirmed to be nonpathological on long-term observations. The pattern of GTT presented here has been found frequently not only in the examinees presently employed but also in those found in the epidemiological studies so far carried out [8, 91. Therefore, it is considered that this pattern of GTT is frequent among the Japanese population. It has been repeatedly pointed out that the clinical manifestation of diabetes mellitus is largely dependent upon races and environmental factors. It was also reported that values in GTT or IRI responses are different among races [23, 241. Accordingly, it seems reasonable to consider that the GTT pattern, as in the case with the clinical manifestation of diabetes mellitus, is somewhat different among the Japanese people from that among the Western people. Such consideration may satisfactorily explain epidemiological features of diabetes mellitus among the Japanese [8,9]. On the other hand, as observed in the follow-up test, the rate of subsequent deterioration of glucose tolerance increased markedly when the value of the initial test exceeded a critical level. Therefore, it is also reasonable to establish criteria for the detection of such cases as early as possible. Thus, the ‘predictive criteria’ were proposed presently. Most of the criteria proposed so far were directed to this intention. I

a

Blood glucose FIG. 5. Conceptional

distribution

level,

mg / 100ml

of normal and abnormal criteria.

groups and two different

diagnostic

The values obtained from this viewpoint, however, indicate some discrepancy from the normal range obtained from the statistical observation of normal subjects. But such discrepancy can be explained as illustrated in Fig. 5, where the plain distribution

138

AKIRA SASAKI and NARUTO HORIUCHI

curve refers to ‘normal subjects’ and the shadowed distribution ‘diabetics’ including those who have a risk of developing diabetes in the future. That is, in covering those with a possible diabetic risk, point a would provide the best separating point, while for an assumption that 98 percentile (mean + 2 S.D.) of the distribution of normal subjects would be the upper limit of the normal range, the diagnostic standard should take point b. As evident in the figure, the line drawn at point a would result in a high ‘false positive’ rate, i.e. a probability of making a misdiagnosis of diabetes on normal subjects, and accordingly a very low specificity. On the other hand, since in establishing point b, the distribution of diabetics was not taken into consideration, it could not be avoided to some extent to overlook those with a risk of developing diabetes mellitus. After all, these two point a and b were established from different standpoints, one from a high specificity in diagnosis and another from a high sensitivity. Therefore, as obvious in Fig. 5, it would be very difficult to establish a single criterion which fits both purposes without a risk of overlooking or misdiagnosing normal subjects as diabetics in a considerable degree. Murphy et al. [20] introduced the idea of a cost function in such a delicate problem of diagnostics. That is, since the costs of false positive and false negative in general are not equal, their weighing should be taken into account. For example, as they indicated, diagnostic criteria for mass chest X-ray for active tuberculosis should be set low, because overlooked cases are very costly both from the standpoints of prevention and treatment. But the usual cost of false positive is small; nothing more than brief anxiety on further examination. In contrast, in diagnosing multiple myelomatosis, the interpretation of the laboratory test, e.g. the plasma protein level, should be very careful, and the prognosis is hopeless. In such a case, diagnostic criteria should be set high being followed by careful clinical observations. In diabetes mellitus among the Japanese, the disease is generally mild, mortality is low and severe cardiovascular complications are rather few. Thus it would be practical to set diagnostic criteria as high as possible without being afraid of overlooked cases. For since a normal group and a diabetic group are supposed to be distributed as illustrated in Fig. 5, in epidemiological studies or health checkup situations, too strict diagnostic criteria would just result in a large number of false positive cases; a great many healthy cases with no fear of possible development of diabetes would be forced to dietary or other treatment. But at the same time, from the standpoint of prevention it is to be hoped that those whose blood glucose level exceeded a certain value were very likely to show further worsening of glucose tolerance. Therefore, those subjects, who are not necessarily classified as diabetics, ought to be observed for a certain period with repeated examination for an early detection of diabetic status. SUMMARY In an attempt to establish diagnostic criteria for diabetes mellitus applicable to the Japanese people, a study was made of distribution of normal blood glucose values on subjects admitted for a multiphasic health examination at the Center for Adult Diseases, Osaka. Five hundred and eighty-six subjects were selected who were without abnormal values on certain standards for various examinations. (1) On an assumption

Studies on Normal Blood Glucose Level

139

that blood glucose values are distributed normally, it was tried to exclude an abnormal group, and a ‘normal range’, i.e. 98 percentile of the distribution, was computed for each age group. (2) The normal range thus obtained showed that the levels in fasting and at three hours were equal to or rather lower than the so far accepted criteria and that the l- or 2-hr level, 180-190 mg/lOO ml, and 140-150 mg/lOO ml, respectively, was considerably higher, showing an increase as age advanced. (3) A follow-up examination of 272 subjects was performed five to seven yr after the initial test to confirm the above values. The blood glucose level in GTT varied rather widely, but in the same trend with the regression phenomenon. The upper limits of the normal blood glucose level estimated presently were included in this range of variation. This fact means that these levels are not abnormal but within a range of normal variation. (4) The number of subjects showing deterioration of glucose tolerance during the seven yr follow-up period increased markedly when the value of the initial test exceeded 110 mg/lOO ml fasting, and 160, 120, and 110 mg/lOO ml at 1, 2 and 3 hr respectively. Therefore, these figures were considered to be on a predictive diagnostic level. REFERENCES I. 2. 3. 4. 5.

10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20.

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AKIRA SA~AKIand NARUTOHORIUCHI Camerini-Davalos R, Caulfield JB, Rees SB, Lozano-Castaneda 0, Naldjian S, Marble A: Preliminary observation on subjects with prediabetes. Diabetes 12: 508, 1963 Jackson WPU: Present status of prediabetes. Diabetes 9: 373, 1960 Rimoin DL: Ethnic variability in glucose tolerance and insulin secretion. Arch Intern Med 124: 695, 1969 Dales LG, Siegelaub AB, Feldman R, Friedman GD. Seltzer CC, Cohen MF: Racial difference in serum and urine glucose after glucose challenge. Diabetes 23 : 327, 1974 Fajans SS, Conn JW: An approach to the prediction of diabetes mellitus by modification of the glucose tolerance test with cortisone. Diabetes 3: 296, 1954