Sigvald B. Refsum

Studies on the Metabolic Error in Refsum's Disease*

Studies utilizing mevalonic acid-2-(14)C and D(2)O as precursors failed to provide evidence for an appreciable rate of endogenous biosynthesis of phytanic acid in a patient with Refsums disease. Orally administered tracer doses of phytol-U-(14)C were well absorbed both by seven normal control subjects (61 to 94%) and by two patients with Refsums disease (74 and 80%). The fraction of the absorbed dose converted to (14)CO(2) in 12 hours was 3.5 and 5.8% in Refsums disease patients and averaged 20.9% in seven control subjects. Labeled phytanic acid was demonstrated in the plasma of both control subjects and patients given phytol-U-(14)C, establishing phytol in the diet as a potential precursor of phytanic acid. This labeled phytanic acid had disappeared almost completely from the plasma of the seven control subjects by 24 to 48 hours, whereas it persisted at high concentrations in the plasma of the two patients for many days. We conclude that the phytanic acid accumulating in Refsums disease is primarily of exogenous origin and that patients with Refsums disease have a relative block in the degradation of phytanic acid and possibly other similar branched-chain compounds. This may relate to a deficiency in mechanisms for release of phytanic acid from stored ester forms or, more probably, to reactions essential to oxidative degradation of the carbon skeleton.

Clinical and biochemical heterogeneity in conditions with phytanic acid accumulation

Phytanic acid accumulation has for more than 20 years been used as a diagnostic criterion of Refsums disease. Recently, however, phytanic acid has also been found in peroxisomal disorders (Zellwegers syndrome, neonatal adrenoleukodystrophy, infantile Refsums syndrome, rhizomelic chondrodysplasia punctata). The 17 patients with Refsums disease in the present study had serum phytanic acid values differing from 73 to less than 0.5 mg/dl (normal). alpha-Oxidation of phytanic acid in skin fibroblast cultures showed a defective capacity in all, with only small differences in residual activity. Phytanic acid determinations in serum from 3 of the 7 patients with peroxisomal disorders showed slightly elevated levels in 2. The alpha-oxidation capacity in the fibroblasts was defective in all, with a residual activity similar to that of Refsums disease. An assay of the alpha-oxidation capacity may be useful in the diagnosis of both Refsums disease and the peroxisomal disorders. The distinction between Refsums disease and the peroxisomal disorders can easily be done on a clinical basis.

Cell renewal of the rat corneal epithelium: A method to compare corresponding corneal areas from individual animals

Abstract A stathmokinetic method to study the mitotic rate in the corneal epithelium of the rat is described and applied. Horizontal and vertical diameter sections were used. In order to analyze the mitotic rate in the various parts of the corneal epithelium, each vision field (objective 100, eye piece 12.5), comprising 182 μm basement membrane, was regarded as a separate unit. Since the number of vision fields acros the cornea varies from specimen to specimen, we constructed a mathematical method to correlate corresponding corneal areas from different eyes. Using this method it is shown that the mitotic rate is almost equal all over the cornea, with no definite reduction in the central areas. There is no area of significantly high proliferation rate, either in the limbal area or in the adjacent conjunctiva.

Circadian variation in the mitotic rate of the rat corneal epithelium: cell divisions and migration are analyzed by a mathematical model

SummaryA stathmokinetic method was used to study the diurnal variation in the mitotic rate (MR) of the rat corneal epithelium, and in the adjacent conjunctival epithelium. A prominent circadian variation in cell proliferation was observed in both epithelia, both showing almost the same pattern, which may indicate that both tissues are submitted to the same regulatory mechanisms. The average rate of cell renewal during a 24 h period indicated a mean cell renewal time of 12.3 days. This is longer than previously assumed. The MR declined toward the central cornea. Based on the above observations and the known centripetal migration of cells in the corneal epithelium, we have developed a mathematical model showing isomorphism with the renewal of the corneal epithelium.

Cell kinetics during healing of corneal epithelial wounds

Abstract. After removing a circular area of the central corneal epithelium of the rat eye, the labelling indices and the mitotic rates were measured at various times after wounding, both in the cornea and in the adjacent conjunctival epithelium. The proliferative response was most marked in the corneal epithelium adjacent to the wound, but there was also a definite response in the epithelium covering the denuded areas, and in the conjunctival epithelium. The study demonstrated that the conjunctiva itself plays a role in the healing of a central corneal epithelial wound. The similarities in the cellular response may indicate that both epithelia are under the influence of the same growth‐suppressing factors (chalones), and must be looked upon as a unit. However, no support was found for the theory that the limbal area serves as a generative organ for the corneal epithelium.

Cell kinetics of conjunctival and corneal epithelium during regeneration of different-sized corneal epithelial defects.

Abstract. Rats with small (diam. 1.7 mm), medium sized (diam. 35 mm) or large (diam. 5.5 mm) corneal epithelial erosions in one eye were killed 1,2 or 4 days after the injury. The proliferative response was evaluated by measuring the labelling index and the mitotic rate in the corneal epithelium and in the adjacent conjunctiva. The small erosions triggered a proliferative response in the cornea only with the maximum response occurring midperipherally. The medium sized erosions induced a higher and more extensive response in the cornea and also a slight increase of the labelling index in the limbal area. The large erosions induced an even more pronounced response in the peripheral cornea and an increase both of the labelling index and the mitotic rate well beyond the limbal part of the conjunctiva. It is concluded that the magnitude and the extent both of the conjunctival and the corneal regenerative response to a corneal abrasion is correlated to the size of the corneal defect. Temporary reduction in the conjunctival epithelial cell number shows that both cells in the limbal and the extralimbal conjunctiva migrate centri‐petally during healing of large corneal wounds. It is suggested that the stem cell theory should be modified. The limbal area is probably an area in which conjunctival epithelial cells or conjunctiva‐derivated cells transform or differentiate to corneal epithelial cells.

Long-Term Endocrine Function of Duct-Ligated Pancreas Isotransplants in Rats

Duct-ligated pancreas transplants were used to study the long-term B cell function after total acinar atrophy. Vascularized whole-organ pancreas transplantation was performed in 47 streptozotocin-diabetic inbred Wistar rats. Of 31 recipients which survived the first week, 30 were permanently cured of the diabetic state with restoration of normal blood glucose levels within 24 h. Metabolic and morphologic studies were performed for up to 16 months after transplantation, i.e. for most of the normal life span of the rats. The recipients of duct-ligated, heterotopic transplants demonstrated plasma insulin (IRI) values slightly above normal. Median blood glucose values were significantly lower than in the normal controls. Basal and stimulated IRI as well as glucose tolerance tests failed to reveal any reduction in the endocrine capacity of the transplants as compared to nondiabetic control. Light-microscopic examinations of grafts showed total acinar atrophy after the first weeks of duct occlusion. No apparent reduction of islet tissue was noted. The results demonstrate that total occlusion of the exocrine ductal system does not impair B cell function of the rat pancreas. The duct-ligated pancreatic transplant permanently reverses induced diabetes when pancreatitis and immunologic reactions are avoided.

Time Dependency in the Regenerative Response to Injury of the Rat Corneal Epithelium

In the present study a central corneal epithelial defect (diameter 3.5 mm) was made in both eyes at 12:00 h in one group of rats and at 24:00 h in another group to see if the regenerative proliferation is influenced by circadian rhythms. The labeling index and the mitotic rate were registered at 4-h intervals in the perilimbal conjunctiva, the limbal area, and different parts of the cornea from the following morning until noon the day after that. The most pronounced regenerative proliferation was seen in the midperipheral and peripheral cornea. The regenerative response occurred in both groups 24-28 h after the injury, but was highly influenced by the normal circadian rhythms, especially with regard to the mitotic rate. The results support the theory that even regeneration is influenced by a circadian proliferative factor.

The Labelling Index Is Not Always Reliable. Discrepancies Between the Labelling Index and the Mitotic Rate In the Rat Corneal Epithelium After Intraperitoneal and Topical Administration of Tritiated Thymidine and Colcemid

Abstract. Many cell kinetic studies are based on the assumption that tritiated thymidine injected into an animal is available for incorporation into DNA for only a short time, and that it labels all cells in the S phase. the present study indicates that this is not the fact for the rat corneal epithelium. the labelling index (LI) declines considerably from the limbal area to the central cornea, while the mitotic rate is almost constant all over the corneal epithelium. the LI should therefore not be used as the only criterion in the assessment of proliferation rate.

Circadian variation in cell proliferation and maturation. A hypothesis for the growth regulation of the rat corneal epithelium.

SummaryThe rat corneal epithelium has been chosen as a model for studying growth regulation. In this epithelium a large single cohort of cells enters the S phase during a fairly short time period once a day. The factor responsible for this wave of cell proliferation is unknown, but it may be a chemical signal from the central nervous system (the suprachiasmatic nucleus or the corpus pineale). The mature cell compartment of the corneal epithelium is assumed to produce a negative feedback factor (chalone), counteracting the effect of the circadian proliferative factor on the local cell proliferation. When no circadian factor is being produced, during most of the 24 h, the chalone seems to enhance the maturation process. During diminished chalone production (e.g. after cell injury and subsequent regeneration), we will get a more or less unrestricted cell proliferation in the tissue with a delayed maturation process prolonging the chalone depletion. This interaction between the circadian proliferative factor and the negative feedback factor for regulation of proliferation with its accompanying stimulatory effect on maturation, may represent a general mechanism in the regulation of cell proliferation in any tissue. Since in at least some organs virtually all cells entering the S phase do this as a single wave once a day, this mechanism may be enough to explain the regulation of cell proliferation during both normal and regenerative conditions.