[1] 122w tions are not precise enough to ascertain that there is a day-night rhythm for this catecholamine. A 24-hour rhythm in norepinephrine turnover in nerves innervating the pineal gland probably reflects diurnal varia- tions in the release of the neurotrans- mitter. The daily rhythm in stimulation by norepinephrine of /3-adrenergic receptors on pineal cells appears to be responsible for the circadian cycle in pineal indoleamine metabolism. That the rhythmic changes in pineal indole- amines persist in blinded rats but can be abolished by interrupting nerve impulses from the brain to the superior cervical ganglia suggests the presence of a "clock" in the central nervous system of the rat. Recent work (21) suggests that this clock resides in the suprachiasmatic nucleus of the hypothalamus

[1] 136w Figure 1 illustrates the extensive moditication seen in erythrocytes from the circulating blood of Duchenne dystrophic patients and carriers. Figure la shows the biconcave character of nor- mal erythrocytes which have not been subjected to a saline wash. After two such washes, cells from normal indi- viduals become somewhat flattened but still maintain their disklike shape (Fig. lb). In contrast, a high percentage of cells from individuals with Duchenne muscular dystrophy are drastically de- formed with many surface projections. This "echinocyte" configuration (8) is also observed with unwashed cells but is more pronounced in cells that have been subjected to the stress of saline washing (Fig. ld). In such washed cell samples from dystrophic patients, the number of distorted cells approaches 100 percent, while samples from nor- mal individuals contain no more than about 7 percent.

[2] 111w Table 1 is a summary of the results of scanning electron microscopic ex- amination of blood from normal indi- viduals, probable carriers, and patients exhibiting several clinical categories of dystrophy. In all instances, clinical dystrophy is associated with an elevated proportion of distorted cells. Since the distortion is visible under a light micro- scope when unfixed cells are diluted with saline solution and examined at a magnification of 450, alterations in the surface do not appear to reflect damage from fixation or coating. The higher resolution obtainable with scan- ning electron optics permits detection of intermediate degrees of distortion and thus a more accurate measure of the proportion of altered cells.

[3] 143w Reliable identification of carriers of the Duchenne form of dystrophy has proved difficult. Thus, it is of interest that deformed erythrocytes are also ob- served in the blood of probable carriers, being present in a proportion generally intermediate between that of dystrophic patients and that of normal individuals (see Table 1 and Fig. Ic). Mothers of dystrophic offspring who are likely car- riers are identified on the basis of a his- tory of the disease in the family and elevated serum concentrations of such enzymes as creatine phosphokinase (CPK). One mother of a dystrophic child (not included in Table 1) was found to have normal erythrocytes; she had neither a familial history of the disease nor elevated CPK activity. The occurrence of dystrophy in her child is thus probably the result of mutation, such events giving rise to about one-third of Duchenne cases.

[1] 46w Abstract. Erythrocytes from patients with congenital muscular dystrophy ex- hibit dramatic surface deformation when observed with a scanning electron mi- croscope. A similar alteration, but one affecting a smaller proportion of cells, oc- curs in the case of female carriers of the sex-linked Duchenne dystrophic condition.

[2] 15w These observed changes in the erythrocyte surface may reflect a systemic defect in membrane properties.

[3] 149w The fundamental lesion underlying congenital muscular dystrophy has been variously suggested to reflect a purely myopathic (1), neuronal (2), vascular (3), or autoimmune (4) mechanism. Investigations of the congenital disease in the laboratory mouse have provided evidence that effects of the lesion are systemic and are, moreover, discernible in altered membrane properties, includ- ing permeability to cations by liver mitochondria (5) and structural irregularities in the surface of erythrocytes (6). It is possible to question the com- parability of the mouse disease, which is autosomal, to human Duchenne mus- cular dystrophy, which is sex-linked (7); it is therefore desirable to examine the human disease, as well, for signs of possible membrane alteration. We report here the results of a scanning electron microscopic examination of erythrocytes from patients suffering from sev- eral categories of human muscular dys- trophy as well as erythrocytes obtained from carriers of the sex-linked (Duchenne) form.

[4] 245w Samples of blood were donated at the muscular dystrophy clinic at the Milton S. Hershey Medical Center by patients, their normal siblings and parents, and laboratory personnel. Blood was ob- tained by a finger stab and drawn into a heparinized capillary tube. A 10-,ul portion was diluted tenfold in cold 0.9 percent NaCl and centrifuged at 900g for 3 minutes. Sedimented cells were resuspended in 100 td of the same medium and centrifuged again. The sedimented cells were suspended in 3 percent glutaraldehyde containing 10 mM sodium cacodylate buffer (pH 7.4) and incubated for 2 hours at 22°C. Cells were then centrifuged, washed in 0.9 percent NaCl, and dehydrated in 70 percent ethanol and, after 5 minutes, 95 percent ethanol. Cells were finally diluted with 95 percent ethanol, spread on glass microscope slides, and dried in air. (In other samples, critical-point drying was employed, and this had no effcct on the observations.) The slides were cut into 1-cm squares and glued to aluminum stubs, shadowed to a depth of 200 A with gold-palladium (60 per- cent-40 percent), and examined in an AMR-900 scanning electron microscope at 21 kv and 300 tilt. Three areas on each stub were selected at random and photographed at a magnification of about 600, and cell counts were made directly from such photographs. In some instances, the two initial washings with NaCl were omitted (Fig. la) and the cells were immediately fixed in glutar- aldehyde, dehydrated, and coated as described above.

[5] 182w Thus, it appears that the erythrocyte surface is significantly altered in cases of human muscular dystrophy, an ob- servation that both strengthens the anal- ogy between the human and murine dystrophic condition and supports the view that the diseases are associated with systemic changes in membranes. The observed red cell alterations are similar to those associated with a num- ber of clinical conditions, including se- vere liver disease, splenectomy, uremia, abnormal hemoglobin (beta thalas- semia), and congenital deficiency of serum beta lipoprotein (8). In no in- stance is there evidence of a correlation between the presence of any of these conditions and congenital muscular dys- trophy (9). The morphological changes described here are similar to those observed in vitro when normal cells are incubated with fatty acids or lysophosphatides (8). Red cell membranes from patients with muscular dystrophy have been shown to differ from normal ones with respect to fatty acid and phospholipid compo- sition (10), with, for example, an in- crease in sphingomyelin. Thus, it is likely that the observed morphological changes reflect alterations in the lipid component of the membrane.

[6] 68w Finally, since treatment of normal erythrocytes with calcium at a high pH leads to similar transformed surface morphology (11), and since we have already observed altered cation movement in liver mitochondria from dys- trophic mice (5), we are attracted to the possibility that changes in mem- brane lipid produce an abnormal pat- tern of cation distribution which, in turn, is responsible for the dramatic membrane distortion described here.