26-Hydroxylation of 5j6-cholestane-3a,7a,12a-triol and other C2rsteroids was demonstrated in cultured skin fibroblasts from healthy individuals. Activities in skin fibroblasts were -5-10% of those previously found in human liver homogenates, and were inhibited by CO. The apparent K., was lowest for 5,B-cholestane3a,7a,12a-triol (1.3 Mmol/liter) and highest for 5-cholestene-3(3,7a-diol (12 Mmol/liter). The rate of 26-hydroxylation was highest with 7a-hydroxy4-cholesten-3-one. These characteristics are similar to those of hepatic mitochondrial C2rsteroid 26-hydroxylase.In skin fibroblasts from three patients with cerebrotendinous xanthomatosis (CTrX), 26-hydroxylation of C2rsteroids proceeded at a rate of only 0.2-2.5% of healthy controls. No accumulation of endogenous 5,B-cholestane-3a,7a,12a-triol could be demonstrated in these cells, and the lowered formation of radioactive, 26-hydroxylated products could not be explained by dilution of the labeled exogenous substrate.The present results add strong evidence to the concept that the primary metabolic defect in CTX is a deficiency of Cr-steroid 26-hydroxylase.

Cultured fibroblasts were shown to be capable of catalyzing the conversion of 7a-hydroxy-cholesterol to 7a-hydroxy4-cholesten-3-one, an important reaction in bile acid synthesis. The apparent K. was 7 ,umol/liter and V.,,. varied between 3 and 9 nmol/mg protein per h under the assay conditions used.The assay was used to investigate fibroblasts from a patient who presented with a familial giant cell hepatitis and who was found to excrete the monosulfates of 3,j,7a-dihydroxy-5-cholenoic acid and 3,8,7a,12a-trihydroxy-5-cholenoic acid in urine

A mixture of 7a-3H-and 4-'4C-labeled cholesterol was administered intravenously to rats. Cholestanol with 20-30% lower ratio between 3H and '4C than in cholesterol could be isolated from different organs. In a healthy human control, cholestanol isolated from feces had a 3H/'4C ratio which was 28% lower than in administered cholesterol. Cholesterol and coprostanol reisolated in these experiments had the same ratio between 3H and 14C as in the precursor. A previously unknown pathway for formation of cholestanol, involving 7a-hydroxylated intermediates, may explain these results. Under normal conditions, this pathway is responsible for at most 30% of the cholestanol synthesized from cholesterol.Intravenous administration of the 7a-3H-and 4-'4C-labeled cholesterol to a patient with cerebrotendinous xanthomatosis (CTX) resulted in formation of cholestanol which had 70-75% lower 3H/'4C ratio. It is concluded that the novel pathway involving 7a-hydroxylated intermediates is accelerated in patients with CTX. This acceleration may contribute essentially to the accumulation of cholestanol, which is a predominant feature of this disease.7a-Hydroxycholesterol and 7a-hydroxy4-cholesten-3-one might be intermediates in the novel pathway to cholestanol. After intravenous administration of 7_-3H-labeled 7a-hydroxycholesterol in a patient with CTX, significant amounts of 3H were incorporated into plasma and fecal cholestanol. Only small amounts of 7a-hydroxycholesterol and 7a-hydroxy-4-cholesten-3-one are excreted into the intestine, and we therefore conclude that the 7a-dehydroxylation step mainly occurs in the liver. In CTFX, the synthesis of cholestanol may be accelerated because the concentrations of 7a-hydroxylated bile acid intermediates in the liver are increased. A possible mechanism for the conversion of a minor fraction of 7a-hydroxycholesterol into cholestanol is suggested.

Evidence was recently presented that an essential part of the accumulation of cholestanol in patients with cerebrotendinous xanthomatosis is due to acceleration of a novel pathway, involving 7 alpha-hydroxylated intermediates in bile acid biosynthesis as precursors (J. Clin. Invest. 1985; 75:448-456). Such intermediates accumulate in patients with cerebrotendinous xanthomatosis due to lack of the mitochondrial 26-hydroxylase involved in the major pathway for bile acid biosynthesis. The new pathway may involve the following steps: 7 alpha-hydroxycholesterol----7 alpha-hydroxy-4-cholesten-3-one----cholesta-4,6- dien-3-one----4-cholesten-3-one----cholestanol. Accurate methods have been developed for assay of 7 alpha-hydroxy-4-cholesten-3-one and cholesta-4,6-dien-3-one in serum, based on isotope dilution-mass spectrometry. The serum levels of 7 alpha-hydroxy-4-cholesten-3-one as well as those of cholesta-4,6-dien-3-one were found to be markedly elevated in the three patients with cerebrotendinous xanthomatosis. Treatment of two of the patients with chenodeoxycholic acid reduced the serum levels of the two steroids by more than 80%. The concentration of cholestanol was reduced by 72% in one patient and by 48% in the other. The possibility is discussed that accumulation of cholestanol in patients with cerebrotendinous xanthomatosis is secondary to accumulation of 7 alpha-hydroxy-4-cholesten-3-one and cholesta-4,6-dien-3-one.