Donald E. McMillan

Effect of Prolonged Bed Rest on Bone Mineral

Abstract Three healthy adult males were restricted to complete bed rest for periods of 30–36 weeks. Urinary calcium excretion was elevated throughout bed rest, averaging 61 mg./day above the base-line value of 193 mg./day. Maximum urine calcium excretion occurred during the seventh week and was 136 mg./day above the base-line value. Fecal calcium excretion was also increased during bed rest. Sweat calcium was unchanged and represented only 2 per cent of calcium output. Mean calcium balances for the three subjects during bed rest were −202, −207, and −254 mg./day. The measured calcium loss during the entire bed rest period averaged 4.2 per cent of the estimated total body calcium. Calcium balance became more normal but remained negative during the three-week period of reambulation. Phosphorus excreted in the urine and phosphorus balance patterns were similar to calcium patterns. Serum calcium and phosphorus levels did not change appreciably during bed rest, but both levels fell during reambulation. Urinary hydroxyproline and pyrophosphate were mildly elevated during bed rest and fell with reambulation. Gamma ray transmission scanning of the os calcis revealed large losses of mineral during bed rest. The decreased mass in the central portion of this bone ranged from 25 per cent to 45 per cent. Mineral reaccumulated in the central os calcis following reambulation at a rate similar to its rate of loss during bed rest. Bone dissolution during bed rest may occur to a greater extent in weight-bearing bones than in the remainder of the skeleton, and the process appears to be reversible.

Reduced Erythrocyte Deformability in Diabetes

The flow properties of individual erythrocytes have been studied in glass micropipets 4 μ internal diameter. The pressure gradient required to establish a standard oscillatory movement over a 130-μ path in a three-second time period was measured in paired studies comparing diabetic and control erythrocytes suspended in Ringer solution. The pressure requirement was regularly elevated for the diabetic erythrocytes, averaging 50 per cent greater than the controls. Studies of erythrocytes comparing alloxan-diabetic rats with control rats demonstrated a similar elevation in required pressure. Red cells from subjects with hereditary spherocytosis offered less flow resistance than diabetic cells, and red cells from rheumatoid arthritics required no pressure increment. When erythrocytes are ejected from a 4-μ micropipet they return quickly to a discoid shape using stored elastic energy. Slow motion photography revealed that diabetic erythrocytes restore their shape less rapidly than nondiabetic erythrocytes, indicating that their reduced deformability is due to an elevation of either intraerythrocyte or membrane viscosity rather than to increased resistance to bending. Diabetes is regularly associated with an increased intracellular hemoglobin Alc; it is possible that hemoglobin Ale could raise intraerythrocyte viscosity. The observed disturbance in flow properties of individual erythrocytes is subtle. It would affect the flow of blood, particularly through active muscle, and modify the pressure exerted by individual erythrocytes on the muscle capillary wall.

Deterioration of the Microcirculation in Diabetes

Studies of the microcirculation in diabetes in the last fifteen years have concentrated heavily on anatomic and biochemical abnormalities of the capillary basement membrane. Greater insights into basement membrane changes have eclipsed the previous picture of widespread progressive deterioration of the entire microcirculation. The history, variety of organ involvement, pattern of circulatory decline, and associated anatomic, physiologic, and biochemical findings are re-examined so that recently described potential mechanisms for the development of diabetic microangiopathy may be understood in a broader perspective. The possible contributions of seven categories of diabetic changes to damage of the microcirculation are outlined. The categories are: (1) altered basement membrane, (2) altered cellular function, (3) cell metabolic changes, (4) altered blood flow properties, (5) distrubed hemostasis, (6) altered oxygen transport, and (7) altered hormone production. The variety of clinical manifestations in long-standing diabetes related to microangiopathy appears to be due to a combination of a widely variable over-all rate of progression and a differing ability of body tissues and organs to accommodate to the sequential circulatory changes. The slow rate of deterioration in most diabetics suggests that several abnormalities must interact to produce the observed progression. A clear understanding of the interactions responsible for diabetic microangiopathy is becoming more important as new options in the management of diabetes become available.

The Effect of Diabetes on Blood Flow Properties

Blood flow is a complex process combining fluid shearing in both plasma and the interior of red blood cells with elastic deformation of bloods solid elements. The red cell membrane is the major solid in blood, but platelets and white cells contribute solid behavior as well. Changes in bloods flow properties are often hidden by bloods ability to change its pattern of response. Capillary viscometry can be used to examine serum, plasma, and hemoglobin solutions directly, but rotational viscometry, where regularity of fluid shearing can be controlled to a narrow shear rate range, is required to study blood flow effectively. The most striking diabetic blood flow abnormality is best seen in time-based rotational viscometer studies that demonstrate the pattern of development of shear stress as flow becomes established. In such studies blood demonstrates both viscoelastic and thixotropic behavior; in diabetes bloods thixotropy is substantially increased. The diabetic pattern appears to be produced by a combination of reduced erythrocyte deformability and increased erythrocyte aggregation due to plasma protein changes. The plasma protein changes observed in diabetes are linked to the development of glucose intolerance but they are not specific to diabetes. The combined increase in aggregation and resistance to deformation of red blood cells produces blood flow abnormalities that can be detected primarily at low shear rate. Plasma protein changes and alteration in blood viscosity are absent from children with diabetes, while such changes tend to be associated with diabetic microangiopathy in adults. In some in vivo conditions, the effects on blood flow that can be linked to erythrocyte deformability are magnified out of proportion to measurements made in vitro. Vessel wall injury may be produced by the adult diabetic hemorheologic changes, contributing to the development of both diabetic microangiopathy and atherosclerosis.

Erythrocyte Spectrin Glucosylation in Diabetes

Because reduced erythrocyte deformability in diabetes might be mediated by increased noneniymatic glucosylation of membrane proteins, we have isolated and studied the degree of glucosylation of spectrin, the major protein of the inner membrane surface. Spectrin was recovered from the erythrocytes of seven age-and sex-matched diabetic and nondiabetic individuals. The thiobarbituric acid (TBA) method was used to measure the glucosylation of spectrin and of 10 mg of hemoglobin from the same ceils. Increased glucosylation of both spectrin and hemoglobin was found in diabetes. Glucosylation, expressed as a ratio of TBA absorbance to protein weight was nearly three times as high for spectrin as for hemoglobin. The ratio of TBA absorbance to hemoglobin content was examined and found to be curvilinear. A reaction model with both first and second order components was fitted to the data to obtain a reaction constant. When this model and constant were used to adjust TBA ab-sorbances, spectrins glucosylation became less than double that of hemoglobin. Spectrin forms an inner membrane network responsible for returning deformed erythrocytes to their original shape. The reported normal response to stretching of diabetic erythrocytes suggests that increased glucosylation of spectrin fails to stiffen the membrane. Some other mechanism, such as reduced phosphorylation of spectrin and other membrane proteins, may be responsible for reduced deformability of diabetic erythrocytes. But spectrins increased glucosylation might be responsible for the increased number of poorly deformable erythrocytes found among aged diabetic erythrocytes. It may produce this effect by inhibiting the membrane protein transglutamidation responsible for removal of aged erythrocytes.

Elevation of Glycoprotein Fucose in Diabetes Mellitus

The pattern of serum protein-bound carbohydrate elevation in diabetes mellitus has been investigated. Cation exchange chromatography on individual sera was followed by serial separation technics on diabetic and control serum pools. The protein-bound hexose elevation could be accounted for by elevated levels of several serum glycoproteins, especially haptoglobin, α1-acid glycoprotein, and α1-antitrypsin. In contrast, only a small portion of the fucose increase could be accounted for by the elevated glycoprotein levels. Fractionation of the serum proteins revealed the fucose increase to be produced by more than one glycoprotein. When ion exchange and gel filtration chromatography were combined to produce elution areas in which only one glycoprotein could be recognized immunologically, a pattern emerged. Three glycoproteins, whose major common source is the liver parenchymal cell, had increased fucose levels, while three immune globulins, not produced by this cell, had normal fucose levels. The demonstration of increased fucose content only in glycoproteins formed by liver cells suggests a metabolic basis for increased serum protein-bound fucose, and a mechanism relating increased hepatic gluconeogenesis and associated cytoplasmic oxidation-reduction changes to increased fucose synthesis is postulated.

Forearm Skin Capillaries of Diabetic, Potential Diabetic and Nondiabetic Subjects: Changes Seen by Light Microscope

Light microscopic examinations were carried out on sections of skin from thirty diabetic and 101 control subjects. Examination of the periodic acid-Schiff stained material in a blind study revealed an abnormality in sixteen of the thirty diabetics and seven of the 101 controls. The abnormality is a hyalinization of the normally fibrillar area external to the endothelial cell of the dermal capillaries. Among the diabetics, the abnormality was seen more frequently in juvenile diabetes, long-standing diabetes, diabetes with retinopathy, neuropathy, or nephropathy, and in obese diabetics. The incidence in control subjects was not strikingly higher in those with a family history of diabetes (three in thirty-nine) or in acromegaly (one in eleven) than in other control subjects (three in fifty-one). Electron microscopic observations suggest that the lesion may be due to deposition of material in the collagen containing cuff of the capillary wall rather than to basement membrane thickening.

Physical Factors Important in the Development of Atherosclerosis in Diabetes

Circulation of the blood is a process controlled by physical laws. The same physical laws also determine the sites of atherosclerotic plaques. Our understanding of their role in atherogenesis is aided by a review of (1) the difference between a solid and fluid, (2) strain and shear or strain rate and shear rate as describers of motion, (3) the distinction between force as pressure and as shear stress, (4) the effects of viscosity and inertia on fluid motion, and (5) the nonlinear responses of blood and blood vessels. Moving blood affects the vessel wall principally through shear stress, the force due to its viscosity applied parallel to the surface of the vessel. Excessive shear stress occurs near the orifices of branches of the aorta, the arterial areas where atherosclerotic plaques develop. It is produced by a change in blood flow pattern at these sites. Arterial stiffening in diabetes reduces the ability of the branches of the aorta to dilate during systole, a motion that helps to control high wall shear stress. At wall shear stress levels under 400 dynes/cm2, a value only modestly higher than that normally achieved near the orifices of the major arterial branches, the endothelial lining of the arterial wall becomes disrupted, allowing entry of plasma proteins and lipids. Blood viscosity elevation in diabetes, linked to enhanced erythrocyte aggregation and reduced erythrocyte deformability, adds to the shear stress at the vessel wall, favoring increased lipid entry. Exposed to a high lipid influx secondary to excessive wall shear stress, the arterial wall must rely on physical as well as chemical means to remove the lipids. Subendothelial motion generated by diastolic arterial contraction and systolic expansion appears to cause plasmalemmal vesicle formation and dissolution, an event probably important in the removal process. The combination of arterial stiffening and hemorheologic disturbance adds to the burden of increased plasma lipids to favor the development of atherosclerosis in diabetes.

Doublet formation of diabetic erythrocytes as a model of impaired membrane viscous deformation

Erythrocyte deformation involves both viscous dissipation in the cell interior and viscoelastic motion of the cell membrane. Reports that describe reduced filterability of diabetic erythrocytes, altered response to oscillatory motion in a capillary-sized pipet, and impaired packing during centrifugation indicate a disturbance of red cell rheology in diabetes. We have selected conditions that minimize the macromolecule-mediated energy of attraction between erythrocytes and studied erythrocyte motion during doublet formation. Under such conditions, doublet formation frequency is strikingly reduced in diabetes. For nondiabetic erythrocytes the formation rate is 0.73 doublets per minute, whereas for diabetic erythrocytes the rate is 0.23 doublets per minute. In addition, mean velocity of doublet formation was found to be decreased to half of normal in diabetes. Completeness of doublet formation, regularly diminished when cell size of the two component cells was similar, was the same for diabetic and nondiabetic erythrocytes. Observation of several features of doublet formation gave a picture of the mechanical process. The initial cell making contact with the glass microscope slide was observed to remain fixed in position. The late arriving cells ability to form a doublet was seen to decrease rapidly, apparently because it came to adhere to the glass surface. The attractive force between the cells overcomes the force of gravity, but cell deformation resistance slows doublet formation by balancing the tendency for cell-cell contact area to increase. An integral equation combining strain energy and viscous dissipation was applied to the doublet formation process. Slowing of doublet formation in diabetes appears to be produced by a doubling of resistance to rate of change of curvature of diabetic erythrocytes.

Exercise and Diabetic Microangiopathy

Exercise has been advocated in the management of diabetes from time immemorial, and its effects on diabetics have been studied with increasing intensity in recent years. Efforts have been made to examine both its metabolic and physiologic effects. Diabetics develop a disturbance of their microcirculation consisting of anatomic changes in the basement membranes of capillaries and the walls of arterioles and venules. This microangiopathy can produce blindness, slowly developing neuropathic changes, and progressive renal insufficiency. The relationship between exercise and diabetic microangiopathy has not often been examined. In this report, we will review relevant effects of exercise on the diabetic, the effect of established microangiopathy on exercise response, exercise modification appropriate to diabetics with microangiopathy, and the use of exercise in testing for early microangiopathy.