Harris H. Tallan

Mitochondrial abnormalities of liver in primary ornithine transcarbamylase deficiency.

Summary: Deficiency of hepatic ornithine transcarbamylase (EC 2.1.3.3) activity in a 17-month-old female patient is described. Enzyme activity was 11% of the mean control value. Electron microscopic examination of the liver specimen, taken by percutaneous needle biopsy, revealed striking abnormalities of the mitochondria: budlike projections, sausage-link appearance, elongation with short cristae, or the presence of parallel arrays of tubules. These abnormalities do not resemble those seen in Reyes syndrome.Speculation: Abnormalities of the liver mitochondria in patients with ornithine transcarbamylase deficiency have not previously been observed. The specificity of these abnormalities requires study of further cases.

Homocystinuria: Observations on the Biosynthesis of Cystathionine and Homolanthionine

Extract: Patients with homocystinuria and a normal subject excreted cystathionine (1.8–15.0 μmoles/hr) after oral administration of homoserine plus cysteine; administration of both precursors is necessary. Excretion in the urine of the next higher homologue of cystathionine, homolanthionine, was found to occur spontaneously (0.8–1.5 μmoles/hr), but not constantly, in three of seven patients with homocystinuria; loading with homoserine increased homolanthioninuria (2.9–5.7 μmoles/hr).Optimum conditions were established in crude extracts of rat liver for in vitro synthesis of cystathionine from homoserine and cysteine (reverse cystathionase) and of homolanthionine from homoserine and homocysteine. Under these conditions, a greater capacity for synthesis of cystathionine by reverse cystathionase (1856 mμmoles/mg protein/hr \pm 95) than for its cleavage in the forward direction (951 mμmoles/mg protein/hr \pm 26) was demonstrated in liver; brain showed barely measurable activity in either direction. In crude extracts of livers from two patients with homocystinuria, activity of reverse cystathionase (39 and 56 mμmoles/mg protein/hr) was less than cleavage (144 and 396 mμmoles/nig protein hr), and both activities were far less than that found in rat liver extracts under the same conditions.Homolanthionine synthesis in rat brain was almost nil; in the other rat organs, it was far less than cystathionine synthesis by reverse cystathionase (compare 69 mμMoles/mg protein/hr \pm 2 in rat liver); it was greater in extracts of rat liver than in extracts of human liver (6.7–10.8 mμmoles/mg protein/hr in human liver); it was virtually absent from the liver of the vitamin B6-deficient rat, but activity was restored by pyridoxal phosphate added in vitro.Homolanthionine synthesis activity in rat liver was separated from cystathionine synthase by ammonium sulfate fractionation (Table IX); it was not separated from cystathionase by such fractionation or by chromatography on carboxymethylcellulose (Fig. 3).Speculation: Although administration of homoserine and cysteine results in urinary excretion of cystathionine by both normal subjects and those with homocystinuria, it is improbable that this treatment would promote the formation of any considerable amount of cystathionine in the brain, since evidence from rat studies indicates that the necessary

Cystathionine |[beta]|-Synthase Deficiency: Differences in Thermostability between Normal and Abnormal Enzyme from Cultured Human Cells

Summary: The thermostability of cystathionine β-synthase and the effect of pyridoxal phosphate (PLP) and other B6 vitamers on this thermostability were studied in extracts of cultured skin fibro-blasts from normal subjects, from heterozygotes for synthase deficiency, and from patients with homocystinuria due to synthase deficiency.Incubation of crude extracts of normal fibroblasts at 55° (preincubation) for short periods prior to assay consistently resulted in an increase of cystathionine synthase activity (activation) that reached a maximum at 2 min (Fig. 1). Further preincubation resulted in inactivation. Addition of 0.4 mM PLP to the preincubation mixture resulted in greater activation that reached a maximum at 3–5 min. The subsequent inactivation phase proceeded at a slower rate than had occurred in the absence of PLP, resulting in a doubling of the half-life of thermal inactivation at 55°. PLP was the only one of the B6 vitamers to have a protective effect.No significant activation of synthase was observed when extracts of fibroblasts from synthase-deficient patients were preincubated at 55°. Cells from eight patients were studied: extracts from those of four patients demonstrated activity too low to permit measurement of thermostability; extracts of cells from two patients were less thermostable than those from control subjects and showed a protective effect of PLP; extracts of cells from two other patients (sibs) showed very high heat stability in the absence of PLP, but significant inactivation of synthase in the presence of the coenzye (Fig. 2, A and A).Three patterns of response to heating were observed with extracts of fibroblasts from seven obligate hétérozygotes (Fig. 3): 1) no activation; 2) activation with and without prior addition of PLP to the preincubation mixture; and 3) activation only in the presence of PLP. The half-life of inactivation at 55° of the extracts from heterozygotes was similar to that seen in extracts from control subjects.Synthase from cultured long term lymphoid cells also showed activation followed by inactivation when preincubated at 55°, as well as protection by PLP.Speculation: Heat-induced activation of cystathionine synthase is a physical characteristic of the normal enzyme molecule which is altered in enzyme from patients and from some heterozygotes for synthase deficiency. Although in vivo vitamin B6 therapy restores the ability of hepatic synthase from patients to be activated, this restoration appears not to be the result of a direct stabilizing effect of the coenzyme PLP on the apoenzyme molecule. Furthermore, since PLP does not consistently protect enzyme from patients against heat inactivation in vitro, the increase of hepatic synthase activity observed in some patients receiving megavitamin therapy may not be mediated by a protective or stabilizing effect of the coenzyme.

Cystathionase Deficiency: Evidence for Genetic Heterogeneity in Primary Cystathioninuria

Summary: Optimal conditions are described for measurement of cystathionase activity in long term lymphoid cell lines. In 21 control lines established from normal subjects, cystathionase (EC 4.4.1.1) specific activity was 25.8 ± 1.7 (mean ± SE) nmole cysteine/hr/mg protein. Extracts of three lymphoid lines (NB-36, NB-95, and NB-77) established from three vitamin B6-responsive patients with primary cystathioninuria had activity of 3.6–7.3; from a B6-unresponsive patient (NB-68) had no detectable activity; from five obligate heterozygotes for cystathioninuria had activity of 11.2–18.9.Two, or possibly three, different modifications of the cystathionase molecule could be demonstrated in cultured cells from the patients with primary cystathioninuria, based on the effects of added pyridoxal phosphate (PLP) in the enzyme assay, on the extent of reaction with rabbit antihuman hepatic cystathionase, and on the ability to compete with normal lymphoid cell line enzyme extract for the antibody combining sites. When PLP is not added to the assay system, the normal enzyme extract still had 89% of its activity in 1.0 mM PLP; on agar double diffusion analysis it gave a band of identity with normal human hepatic cystathionase; the precipitin band had cystathionase activity, but inhibition by antibody could be shown in solution. Lymphoid line extract from the B6-unresponsive patient had no detectable activity in the absence or presence of PLP, did not form a precipitin band, and did not compete with normal enzyme extract for the antibody combining sites. Thus, synthesis of apocystathionase is absent or significantly reduced; alternatively, the protein produced has lost its antigenic determinants as well as catalytic activity. Extracts of the three cell lines established from the three B6-responsive patients had no activity in the absence of added PLP, but progressively greater activity was found with increasing concentrations of PLP: at 1.0 mM PLP 37, 26, and 16% of the mean normal value, respectively was attained. Cell extract from each of the B6-responsive patients formed a band of identity with normal enzyme on agar double diffusion and cystathionase activity could be demonstrated on the precipitin band only if PLP was added to the extracts before immunodiffusion. Extract of the B6-responsive lymphoid cell line NB-36 combined with the antibody sites for cystathionase, thereby blocking inhibition of normal enzyme, whereas extract of the line NB-95 had only a slight blocking effect and extract of the line NB-77 had no blocking ability. Thus, the lymphoid cell lines from the three B6-responsive patients produce a cyctsthionase with an altered capacity to combine with PLP; the enzyme in lines NB-95 and NB-77 also may have been affected in a manner which changes as well the ability to compete for the antibody combining site. These altered cystathionases, both B6-responsive and B6-unresponsive, also may represent forms more readily degradable than the normal enzyme.Antigenic identity was shown for cystathionase from various organs within a given vertebrate species, but only partial identity was observed among cystathionases from different vertebrate species.Speculation: Primary cystathioninuria is caused by different mutations affecting the cystathionase molecule. One form of cystathioninuria, vitamin B6-unresponsive, appears to result from absence of synthesis of the enzyme protein. B6-responsive forms appear to result from production of cystathionase molecules altered in ability to combine with coenzyme, but with antigenic indentity on agar double diffusion analysis maintained; in some cases, there may be changes in antibody binding capacity as well, although immunologic cross-reactivity remains.

Taurine Availability and Function in Neurogenetic Retinopathies

Several neurogenetic diseases manifest abnormal retinal electrophysiology and pathology; such changes in both retinal integrity and function resemble those found in the retina of individuals with retinitis pigmentosa. Some of these inherited neurodegenerative disease include: ceroid lipofuscinosis, Bardet-Beidl-Lawrence-Moon syndrome and Refsum disease, (36). The etiologies of the retinal degeneration observed in these neurogenetic diseases and retinitis pigmentosa remain unknown. Preliminary results, however, from this laboratory (68) and published reports from other investigators (16,21) suggest altered transport of the amino acid taurine in several neurodegenerative diseases.

METHIONINE-ACTIVATING ENZYME |[lpar]|MAE|[rpar]| DEFICIENCY: A NEW CAUSE FOR HYPERMETHIONINEMIA IN INFANCY

Hypermethioninemia in a neonate, originally found on mass screening, was not accompanied by homocystinemia, cystathioninemia, or hypocystinemia. At 6 months, when plasma methionine was 10-20 times normal, hepatic MAE was Snmoles S-adenosyl-methionine (SAM)/mg prot/h (normal mature MAE = 86 ± 16; normal 2nd trimester fetal MAE =26 ± 3). Activities of cystathionine synthase, cystathionase and N5-methyltetrahydrohydrofolate (Me-THF) homocysteine methyltransferase were normal. “Liver function” tests and a battery of serological tests for hepatogenic infectious agents were normal; the infant appeared healthy. Liver was normal by light microscopy, but electron microscopy showed breaks in the outer membranes of the mitochondria and some hyperplasia of smooth endoplasmic reticulum. Serum folate remained high (>16 ng/ml) 4 months after 15 mg/day for 3 weeks (should clear in 3-4 weeks), suggesting an inability to metabolize Me-THF to THF due to deficiency of SAM, the product of MAE and a cofactor for Me-THF homocysteine methyl transferase. Unequivocal hypersegmentation of polymorphs but no megalo-ovalocytosis of RBC was found. Skin fibroblasts and lymphoid cell lines are growing and will be assayed for MAE. The strikingly low hepatic MAE activity (below fetal) and its persistance to 8 months of age suggests this is a new metabolic disease.

Homocystinuria: Biosynthesis of Cystathionine and Homolanthionine

Cystathioninuria after administration of homoserine and cystein has been confirmed in patients with homocystinuria; both precursors are necessary. Homolanthioninuria has been found to occur spontaneously in two of seven patients with homocystinuria and after homoserine loading in two more. A greater capacity for synthesis of cystathionine from homoserine and cysteine (reverse cystathionase) than for its cleavage (forward cystathionase) was demonstrated in rat liver, kidney and pancreas; rat brain showed very low activity in either direction. Homolanthionine synthesis from homoserine and homocysteine was less than 5% of cystathionine synthesis by reverse cystathionase in rat liver, kidney and pancreas; rat brain showed no activity. In liver extracts from two patients was less than cleavage and both were far less than in rat liver; homolanthionine synthesis activity in rat liver was separated from cystathionine synthase by ammonium sulfate fractionation; it was not separated from cystathionase by such fractionation or by chromatography on carboxymethyl-cellulose. Thus: (1) Although the administration of homoserine and cysteine results in cystathioninuria it is unlikely to promote formation of any considerable amount of cystathionine in the brain. (2) Whatever the excretion of homolanthionine may have to do with the pathogenesis of homocystinuria, it seems unlikely to have anything to do with the dementia. (3) Cystathionase, acting in the reverse direction is probably responsible for the synthesis of homolanthionine.

HOMOCYST INURIA DUE TO CYSTATHIONINE SYNTHASE (CS) DEFICIENCY: INVESTIGATIONS IN CULTURED LONG-TERM LYMPHOCYTES, FETAL SKIN FIBROBLASTS AND AMNIOTIC FLUID CELLS

Systematic delineation of optimal assay conditions for CS in adult skin fibroblasts (fib.) enabled us to differentiate obligate heterozygotes (het.) from affected individuals and controls (BBRC 55:38, 1973). We have now extended our studies to long-term lymphoid lines (LTL) and cultured fib. of fetal origin. We have detected CS activity in LTL from 12 normal donors (mean ± SEM=9.49 ± 0.98 nmoles/mg prot./h), its deficiency in a line from an affected individual (0.88), and intermediate activity in 3 obligate het. lines (3.21 ± 0.37), with no overlap. Activity in skin fib. from 5 control fetuses was 32.9 ± 5.06 and not different from control amniotic fluid (AF) cell cultures (40.7 ± 4.58) (.25>p>.01); both differed from adult skin fib. (21.0 ± 1.71) (p < .001). Activity in AF cells from an obligate het. mother was 73.8; prenatal diagnosis of a normal fetus was confirmed after birth by assay of skin fib. Thus: (1) LTL, which proliferate vigorously in apparently permanent cultures, are ideal for detection and study of the enzymatic defect in homozygotes and het. for CS deficiency. (2) CS values from adult skin fib. cannot be used as controls for AF cells. (3) Requirements for prenatal diagnosis of CS deficiency have now been met. (4) CS activity in het. LTL, as in liver and skin fib., was less than 50% of normal.