Endothelial factors in relation to peripheral circulation in diabetics

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THROMBOSIS RESEARCH 20; 231-238, 1980

ENDOTHELIALFACTORS IN RELATIONTO PERIPHERAL CIRCULATIONIN DIABETICS L.-O. Almgr, B. Lilja, S.E. Lindell,I.M. Nilsson From the Departmentsof InternalMedicine,Clinical Physiologyand CoagulationLaboratory,Universityof Lund, Allmgnna Sjukhuset,S-214 01 Malmt,,Sweden

(Received23.9.1980. Acceptedby Editor H. Stormorken)

ABSTRACT Infrared thermography was used to study the increase in foot temperature of 58 diabetics after immersion of the feet in cold water for 10 minutes. Forty minutes later, after indiregt heating, the temperature increase of the feet was g 3 C in 27 patients (group A). The remaising 31 patients (group B) had a mean increase of lo-12 C in the same time interval. Plethysmography and segmental blood pressure determinations showed no significant differences between the blood flow of the lower extremities in group A and B. The fibrinolytic response to stimulation was abnormally low in 65 % of those in group B compared to 19 % in group A. VIIIR:Ag was significantly higher in group A than in group B. The systolic blood pressure was significantly higher in group A. Hypothetically, the differences between group A and B might be explained by an imbalance of the components of the autonomic nervous system. INTRODUCTION It is a wellknown fact that there are changes in endothelial factors in diabetics (1). Factor VIII related antigen (VIIIR:Ag) which is synthesized in the vascular endothelial cells and is most important for the adhesion of platelets, is frequently Key words: diabetic autonomic neuropathy, fibrinolytic activity, factor VIII, thermography, plethysmography, pathogenesis of diabetic angiopathy

231

232

F. VIII R: AS/PA vs. VASOACTIVITY

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increased (2). Adhesion and aggregation of platelets might cause occlusions of the microcirculation as indicated by fluorescein angiogram (3). On the other hand, the degradation of vascular occlusions by fibrinolysis is often impaired in diabetes. Fibrinolysis is dependent upon another endothelial product, an activator that transforms the inactive plasminogen of the blood into active plasmin. Plasmin then starts to degrade fibrin deposits in the vessels. This is one of the few ways for the body to remove vascular occlusions. An abnormal balance between these factors might contribute to the pathogenesis of diabetic angiopathy. To study the circulation of the lower extremities of diabetics, a combination of thermography (4, 51, plethysmography and segmental blood pressure determinations was used. The aim of the present investigation was to correlate any changes of the circulation with abnormalities of these endothelial factors. MATERIAL AND METHODS The material consisted of 58 diabetics (30 men and 28 women), with a mean age of 49.9 years (range 23-77). The duration of diabetes ranged from 2 to 41 years with a mean of 16.7 years. Thirty had a diagnosis of diabetes before the age of 30. Five patients were on diet only, while 32 were on insulin and another 10 on insulin combined with phenformin. Four took phenformin, one sulfonylurea and six were on a combination of phenformin and sulfonylurea. Collection of blood samples. Blood samples were obtained between 8 and 9 a.m. from fasting patients for blood glucose, which was determined with the glucose oxidase method (6), plasma cholesterol (7) and plasma triglycerides (8). Before samples were obtained for determining the fibrinolytic and coagulation factors described below, the patients requiring insulin, sulfonylurea or biguanide got their usual dose and had breakfast. None received treatment with any other drugs known to be capable of influencing hemostatic variables. The blood was collected in siliconized tubes and titrated plasma was prepared. Spontaneous fibrinolytic activity was measured by testing redissolved euglobulin precipitate of titrated plasma on unheated bovine fibrin plates (9). The mean spontaneous fibrinolytic activity in 153 age and sex matched controls was 34.4 f 3.3 mm2. Fibrinolytic response to venous occlusion. The local fibrinolytic activity developing during artificial venous occlusion of the arms was measured according to Robertson and others (IO). Venous occlusion was produced by placing a sphygmomanometer cuff around the upper arms and inflating it to a pressure midway between the systolic and diastolic blood pressure in the limb for 20 minutes. Blood samples were drawn immediately before the cuff was deflated, and the fibrinolytic activity of re-suspended euglobulin precipitate was determined on unheated fibrin plates. The mean fibrinolytjc activity after such occlusion of both arms was 313.8 f 10.2 mm in 153 age and sex matched healthy controls. Plasminogen activator activity of vein walls. The fibrinolytic activity of biopsy specimen of a superficial dorsal vein of the hand was assessed by a modification (11) of the histochemical method of Todd (12).

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Fibrinogen. Citrated blood was collected with EACA and the sample assayed with a synerese method according to Nilsson & Olow (9). Normal mean 3.0 2 0.06 g/l. a -macroglobulin. Esterolytic method (13). Normal range 80-720 %. Inhibitors of plasminogen activation by urokinase (urokinase inhibitors). Clot method described by Paraskevas et al. (14). Normal range 60-140 8. Factor VIII:C. The biological activity of factor VIII was assessed from its normalizing effect on the recalcification time of platelet-rich hemophilia A plasma. The amount of factor VIII was expressed as percentage of that found for a normal standard (15). Normal range 60-160 %. Factor VIIIR:Ag was quantitatively determined by electrophoresis in agarose gel containing antibodies by the rocket technique of Laurell. The monospecific precipitating antibodies were raised by injecting rabbits with a purified AHF-preparation as described by Holmberg & Nilsson (16). Normal range 60-175 %. The tests for the evaluation of the blood flow of the lower extremities were performed after all the above mentioned blood tests had been taken. The methods used for estimation of the circulation of the legs were venous occlusion plethysmography (17) and determination of the systolic blood pressures of the right arm and great toes measured according to Gundersen (18) with the strain gauge technique. A pressure fall of more than 45 mm Hg from arm to great toe was considered as abnormal. For venous occlusion plethysmography the lower levels of the normal ranges for first flow and maximal flow were set at 14 and 17 ml/100 ml leg volume, respectively (19). Thermography. Equipment: The infrared emission was recorded with Aga Thermovision Equipment System No. 661, which works on a scanning principle. The object is scanned by mirrors and prisms directing the emission to an indium antimonide detector cooled by liquid nitrogen. The optical signal is transformed into electronic pulses which are amplified and modified to give a picture on an oscilloscope screen. The visible picture is then photographed. An isotherm set at various temperature levels is introduced into the infrared picture on the screen in order to obtain an objective measure of differences in emission levels. Methods. The measurements were performed in a room especially devised for infrared thermography with a temperature maintained constantly around 200C. All patients were given ethanol orally to avoid vasoconstriction. The feet were immersed for 10 minutes in water with a temperature of 150C. Then the patients lay down, the feet were wiped off and a heater was placed over the trunk to provide a means of indirect warming of the feet. The infrared emission from the skin of the feet was recorded every 5th minute for 40 minutes. The mean temperature of the toes of each foot was calculated. RESULTS There was a considerable variation of the foot temperature in the patients at rest before cooling but there were no sex differences. After the feet had been immersed for ten minutes in water with a temperature of 150C , all had a mean foottemperature

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of 18-20°C. Indirect warming increased the foot temperature. After 40 minutes the women had reached a significantly (p
I

Some Variables (Means f SD) in Those with an Increase of the Temperature of the Toes of 3oC or Less (Group A) and More (Group 8) after 40 Minutes of Indirect Warming after Initial Cooling A

B

27

31

Age (years)

53.6 + 15.6

47.3 t 14.0

N.S.

Duration of diabetes (years)

15.8 f 7.7

17.4 5 10.1

N.S.

Mean temperatures of the foot before cooling warmest side coldest side

24.3 t 2.7 23.4 + 1.9

26.1 t 3.6 25.1 t 3.3

immediately after cooling warmest side coldest side

18.9 t 0.5 18.8 t 0.5

19.1 f 0.6 18.9 t 0.6

40 min after cooling by indirect heating warmest side coldest side

21.5 + 2.0 20.6 t 0.9

30.7 + 2.5 28.8 ; 3.5

Plethysmography: first flow (ml/100 g tissue/min)

16.2 + 5.7

13.8 t 4.0

N.S.

Plethysmography: maximal flow (ml/100 g tissue/min)

Number of patients

Significance

p <0.05 p (0.02 N.S. N.S.

followed

20.8 + 6.0

19.5 t 3.8

N.S.

Mean systolic blood pressure of great toe; lowest recorded in each patient (mm Hg)

104 + 41

110 + 24

N.S.

Mean systolic blood pressure of right arm (mm Hg)

152 2 29

133 + 22

p
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TABLE

II

Some More Variables (Means + SD) in Those with an Increase of the Temperature of the Toes of 3oC or Less (Group A) and More (Group B) after 40 Minutes of Indirect varming after Initial Cooling A Actual/ideal

weight

Blood cholesterol

1.15

(mg/lOO.ml)

Blood triglycerides

(mMol/l)

Blood glucose (mg/lOO ml) Urine glucose (g/24 h) Fibrinolytic re ponse to venous occlusion (mm 2 lysis) Spontaneous fibrinolytic (mm2 lysis) Fibrinogen

1.13

Significance N.S.

215 + 37.

208 + 41

N. s..

1.6 t 0.7

1.3 + 0.9

N.S.

160 + 69

187 + 81

N.S.

19 + 32

24 + 33

N.S.

268 + 126

153 2 99

p
29 + 24

26 + 25

N.S.

3.3 + 0.8

3.1 + 0.5

N.S.

activity

(g/l)

a2-macroglobulin

B

145 t 38

157 + 60

N.S.

Inhibitors of plasminogen activation (% of normal)

(% of normal)

129 + 30

133 2 39

N.S.

VIIIR:Ag

(% of normal)

173 + 64

140 + 49

p < 0.05

VIII:C

(% of normal)

154 + 51

150 + 60

N.S.

There were no significant differences as regards sex, age, type and duration of diabetes, treatment of diabetes, relative weight, smoking habits, plethysmographic findings (first flow, maximal flow), cholesterol and triglyceride levels or grade of diabetes regulation. Sixteen in each group had ophthalnoscopically visible retinopathy. Ten patients in group A and 13 in group B had signs of peripheral neuropathy. The mean level of systolic toe pressures (lowest recorded on any side) was 104 + 41 mm Hg in group A, as compared to a mean of 110 + 24 mm Hg In group B. However, the mean systolic pressure of the Eight arm was significantly higher (p (0.01) in group A (1522 29 mm Hg) than in group B (133 f 22 mm Hg). Thus, the systolic pressure gradient between the systolic pressure of the arm and the great toe was significantly higher (p
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(p x0.05) in group A (173 2 64 %I than in group B (140 + 49 %). However, the biological activity of factor VIII did not differ between these groups. DISCUSSION To our surprise, those who had a markedly small increase in foot temperature on indirect warming after cooling did not show any signs of impaired blood flow as measured with water plethysmography. On the contrary, the mean first flow and maximal flow were somewhat, although not significantly, higher than in those with a rapid temperature increase after cooling. This should exclude marked stenosis of the main arteries of the legs above the knees. Furthermore, the systolic blood pressures in the great toes were similar in the two groups. Thus, most likely, there were no marked differences of the calibres of the arteries below the knees down to the forefoot. Longstanding peripheral vasospasm, in spite of alcohol ingestion before the start of the test, seems to be the only plausible explanation of the great temperature differences of the two groups. Group A seems to consist of patients who are likely to show vasoactive reactions. This might be due to abnormal activity of the sympathetic nervous system and imbalance of the components of the autonomic nervous system. The increased response of peripheral vessels to cooling could also hypothetically result from impaired sympathetic innervation of these vessels (denervation sensitization) (20-28). The patients in group A have a higher systolic mean blood pressure than those of group B, but this difference is reduced in vessels of small calibres as in the digital arteries of the great toes, probably because of an increased tone in the digital arteries from group A. In spite of indirect warming the mean temperature of the feet increases only a few degress above the starting point after cooling, while the patients of the other group increase the foot temperature considerably. It is interesting to observe that the patients in group A also release larger amounts of some of the substances produced by the endothelium. VIIIR:Ag was significantly higher in the blood of patients in this group and after stimulation with venous occlusion of the arms the release of plasminogen activator to the blood ("fibrinolytic response") was significantly higher than in group B. In a previous study we found that 30 % of 221 diabeti s had a poor fibrinolytic response to venous occlusion (
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vasospasm after cooling, a lower systolic blood pressure, and a high frequency of poor fibrinolytic response to stimulation compared to group A. Thus, very marked differences were noted between diabetics with and without vasoactive reactions as regards the release of the endothelial VIIIR:Ag and plasminogen activator. The differences might be caused by changes in the autonomic nervous system which hypothetically might contribute to the development of diabetic angiopathy in this way. ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Diabetes Association, the Nordic Insulin Foundation, and the Swedish Medical Research Council (Nos 4993 and 00087). REFERENCES 1.

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