Oxidation of pristanic acid in fibroblasts and its application to the diagnosis of peroxisomal beta-oxidation defects.

Pristanic acid oxidation measurements proved a reliable tool for assessing complementation in fused heterokaryons from patients with peroxisomal biogenesis defects. We, therefore, used this method to determine the complementation groups of patients with isolated defects in peroxisomal beta-oxidation. The rate of oxidation of pristanic acid was reduced in affected cell lines from all of the families with inherited defects in peroxisomal beta-oxidation, thus excluding the possibility of a defective acyl CoA oxidase. Complementation analyses indicated that all of the patients belonged to the same complementation group, which corresponded to cell lines with bifunctional protein defects. Phytanic acid oxidation was reduced in fibroblasts from some, but not all, of the patients. Plasma samples were still available from six of the patients. The ratio of pristanic acid to phytanic acid was elevated in all of these samples, as were the levels of saturated very long chain fatty acids (VLCFA). However, the levels of bile acid intermediates, polyenoic VLCFA, and docosahexaenoic acid were abnormal in only some of the samples. Pristanic acid oxidation measurements were helpful in a prenatal assessment for one of the families where previous experience had shown that cellular VLCFA levels were not consistently elevated in affected individuals.

Accidental hypothermia.

Knowledge of the effects of hypothermia has increased greatly over the past 25 yr. Thousands of patients have been cooled intentionally in the operating room, and hundreds of thousands of living hearts have been temporarily stopped by cold cardioplegia and restarted without difficulty or apparent ill-effect. Yet in spite of the acquisition of this vast body of clinical experience an aura of mystery stills surrounds the patient who becomes hypothermic accidentally. The best treatment in any particular case is not always clear, and published accounts do not always give the impression that the hypothermic patient is treated with the same rational approach with which other sick and comatose patients are treated. In summarizing, therefore, conclusions that might be reached from reviewing past experience several important points emerge. The severely hypothermic patient should be treated in an intensive care unit where appropriate monitoring of temperature, cardiovascular function and respiratory function are available, and where full respiratory support including assisted ventilation can be given. The final outcome depends upon the etiology. The young healthy victim of exposure has a good chance of surviving. The patient poisoned by alcohol or barbiturates has a good chance of surviving provided the level of intoxication is not itself lethal. The elderly without severe underlying disease have a good chance of surviving. The patient with severe underlying disease of the endocrine, cardiovascular or neurologic system probably has, at best, a 50% chance of surviving and, at worst, a chance of only 10-20%, depending upon the associated disease. There is no statistical evidence that any one method of rewarming is significantly better than any other. But there is anecdotal evidence that in the absence of full monitoring and support systems slow rewarming is safer than over-energetic external rewarming. Internal rewarming, peritoneal dialysis, hemodialysis, inhalation of warmed oxygen and extracorporeal circulation are effective in severe cases and can be used with safety. The causes of, and triggering mechanism for, ventricular fibrillation are still largely unknown but the onset of ventricular fibrillation in a very cold patient may often be an irreversible complication. The place of modern anti-arrhythmic drugs in the prevention and management of this complication has yet to be elucidated. Cardiopulmonary resuscitation is difficult in profoundly hypothermic patients but should be maintained until a body temperature of 30 degrees C has been achieved.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical localization of sphingolipid activator protein 2 (SAP-2) in normal and SAP-deficient fibroblasts.

The intracellular localization of sphingolipid activator protein 2 (SAP-2) was determined immunocytochemically using an antiserum raised against a SAP-2 preparation from Gaucher spleen. The immunolabeling indicated that SAP-2 was largely localized in the lysosomes of fibroblasts from normal individuals. In some lysosomes the labeling was greatest around the perimeter of the matrix, suggesting an association between the activator and lysosomal membrane components. The labeling technique was also applied to fibroblasts from a patient with a unique sphingolipid storage disorder. Consistent with immunoblotting studies on electrophoretograms, both the patient and his affected fetal sibling were found to be deficient in immunoreactive SAP-2.

Saposins (sap) A and C activate the degradation of galactosylsphingosine.

As previously shown for [(3)H-galactosyl]ceramide, the breakdown of [(3)H-galactosyl]sphingosine was reduced in prosaposin-deficient skin fibroblast homogenates. Galactosylsphingosine hydrolysis was also deficient in cell homogenates from Krabbe's disease (beta-galactocerebrosidase-deficient) patients, but not acid beta-galactosidase-deficient patients. Moreover, hydrolysis of galactosylsphingosine in the prosaposin-deficient cell homogenates could be partially restored by adding pure saposin A or C, thereby identifying these saposins as essential facilitators of galactosylsphingosine hydrolysis. By contrast, saposins B and D had little effect on galactosylsphingosine hydrolysis in the prosaposin-deficient cells. The reduced galactosylsphingosine turnover in prosaposin-deficiency suggests that there could be a pathogenetic cerebral accumulation of galactosylsphingosine in this disorder.

Saposins (sap) A and C activate the degradation of galactosylceramide in living cells.

In loading tests using galactosylceramide which had been labelled with tritium in the ceramide moiety, living skin fibroblast lines derived from the original prosaposin-deficient patients had a markedly reduced capacity to degrade galactosylceramide. The hydrolysis of galactosylceramide could be partially restored in these cells, up to about half the normal rate, by adding pure saposin A, pure saposin C, or a mixture of these saposins to the culture medium. By contrast, saposins B and D had little effect on galactosylceramide hydrolysis in the prosaposin-deficient cells. Cells from beta-galactocerebrosidase-deficient (Krabbe) patients had a relatively high residual galactosylceramide degradation, which was similar to the rate observed for prosaposin-deficient cells in the presence of saposin A or C. An SV40-transformed fibroblast line from the original saposin C-deficient patient, where saposin A is not affected, showed normal degradation of galactosylceramide. The findings support the hypothesis, which was deduced originally from in vitro experiments, that saposins A and C are the in vivo activators of galactosylceramide degradation. Although the results with saposin C-deficient fibroblasts suggest that the presence of only saposin A allows galactosylceramide breakdown to proceed at a normal rate in fibroblasts, it remains to be determined whether saposins A and C can substitute for each other with respect to their effects on galactosylceramide metabolism in the whole organism.

Molecular changes in the D-bifunctional protein cDNA sequence in Australasian patients belonging to the bifunctional protein complementation group.

The cDNA sequence for the human D-bifunctional protein (D-BP: 17 beta-hydroxysteroid dehydrogenase IV) was investigated in patients with peroxisomal disorders belonging to the BP complementation group (CG). In three cases, analysis of polymerase chain reaction products generated from the patients' cDNA indicated the presence of a deletion within the region corresponding to nucleotides 209-537 of the normal cDNA sequence. Subsequent sequencing revealed that, in two of the patients, 47 base pairs were missing, with the deletion corresponding to nucleotides 302/3-349/50 of the normal sequence. In the third patient, a smaller deletion of 22 bp (nucleotides 280/1-302/3) was characterized. Only the mutant sequence was detected in each of these cases, consistent with parental consanguinity. Both deletions cause a frameshift, and would lead to premature termination of the BP. Available family members were also investigated, and the findings conformed with expectations for an autosomal recessive disorder. In addition to the deletions, a number of other base changes have been identified in this series of patients. In particular, one patient, whose parents were also consanguineous, was homozygous for a base change, which results in a nonconservative substitution of serine 177 with a phenylalanine residue. The functional significance of this amino acid substitution, as well as the other identified changes, is still to be determined. Nevertheless, our data provide strong support for the hypothesis that defects in the gene for the D-BP are responsible for the beta-oxidation defect in patients belonging to the BP CG.

Aortic valve replacement with the De Bakey valve.

De Bakey prostheses were inserted in 29 patients with aortic valve disease between October, 1970, and May, 1972. Ten patients have died, but all but one of the remaining 19 have beel followed for a minimum of 19 months. Evaluation of the results in these subjects indicates that the function of the De Bakey valve compares favorably with that of other aortic valve prostheses.

Congenital aortic stenosis: ten to 22 years after valvulotomy.

Between 1956 and 1967, 34 patients, aged 2 months to 40 years, underwent aortic valvulotomy under hypothermia for congenital aortic stenosis. There were two early and five late deaths. Twenty-seven patients were followed up for a mean of 15 years. Thirteen patients had no subsequent operation: 11 are asymptomatic, seven with mild aortic insufficiency. Ten patients have had aortic valve replacement (AVR), one revalvulotomy, three will require AVR. Three late deaths were sudden. The literature has been reviewed for data on mortality, endocarditis, aortic insufficiency, and reoperation. Operation improves longevity, but does not restore it to normal. Aortic valve replacement in children carries a poor prognosis, possibly reflecting severity of disease. The chances of reoperation after ten years are 20% to 40%. Valvulotomy must, therefore, be regarded as the first in a possibly lifelong series of operations.

Pulmonary artery banding for ventricular septal defect with pulmonary hypertension.

Sixty children in this series underwent pulmonary artery banding (PAB); isolated ventricular septal defect (VSD) was present in 24, and 20 were corrected seven months to ten years and eight months after PAB. Other anomalies were present in 37, and 15 were eventually totally corrected. Survival after PAB was 90% (95.8% with isolated VSD), and 94.3% (95% with isolated VSD) after total correction. Mean age at banding was 12.9 months, and 52.6 months at debanding. All patients underwent catheterization before PAB; 39 underwent catheterization after PAB, and ten after total correction. Pulmonary artery blood pressure was reduced from 53.5 mm Hg to 25.3 mm Hg by banding. Most patients had only moderately increased pulmonary blood flow. Banding at the altitude of Denver (1,600 m) appears to be an effective means to convert high-risk infants into lower-risk children for total correction. The cumulative mortality for two-stage treatment of VSD was 8.3%.

Patent ductus arteriosus ligation and respiratory distress syndrome in premature infants.

Ligation of a patent ductus arteriosus was carried out in 22 premature infants, 20 with concomitant respiratory distress. The duration of high-volume shunting is critical in determining the prognosis for these infants. Because of the low surgical mortality and morbidity and the high incidence of bronchopulmonary dysplasia in babies managed conservatively, infants with respiratory distress syndrome (RDS) who are respirator dependent should undergo ligation as soon as the presence of large left-to-right shunting is detetmined. Premature infants without RDS or those with mild RDS who are not respirator dependent can be managed medically or with elective ligation. Surgical intervention is strongly indicated in patients with persistent congestive heart failure and respiratory failure. Echocardiography offers an accurate and risk-free approach to the early diagnosis of a large left-to-right shunt through the ductus.

Model SV40-transformed fibroblast lines for metabolic studies of human prosaposin and acid ceramidase deficiencies.

Skin fibroblasts from patients with Farber disease (acid ceramidase deficiency) and from two siblings of the only known family affected with prosaposin deficiency were transformed by transfection with a plasmid carrying the SV40 large T antigen. The prosaposin-deficient transformed cell lines conserved their original metabolic defects, and in particular they were free of detectable immunoreactivity when using anti-saposin B and anti-saposin C antisera. Ultrastructurally, the cells contained heterogeneous lysosomal storage products. As found for their parental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and beta-galactosylceramidase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radioactive glucosylceramide or low density lipoprotein-associated radiolabelled sphingomyelin by the prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide storage in prosaposin-deficient cells was also demonstrated by ceramide mass determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of acid ceramidase in cells from prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that prosaposin is required for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophysiological studies on human prosaposin deficiency.

An Australasian diagnostic service for the neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCLs) are a family of related genetic disorders that together are believed to affect one child in every 12,500 births in the USA. Our laboratory has developed a diagnostic service for classical late infantile neuronal ceroid lipofuscinosis (LINCL) by assay of tripeptidyl-peptidase I (TPP-I) activity using the fluorogenic peptide substrate Ala-Ala-Phe aminomethylcoumarin, followed by a screen for three mutations in the CLN2 gene. In addition, we have also begun to offer a limited diagnostic service for the juvenile (JNCL) and infantile (INCL) forms of the disease on the basis of mutation analysis of the CLN3 and CLN1 genes, respectively. Retrospective analysis of Australasian patients with a clinical suspicion of NCL has revealed that six are affected by LINCL, six by JNCL and, to date, two by INCL. Mutation analysis of our LINCL patients has shown that the three screened mutations, namely, the nonsense mutation R208X and the splice mutations IVS5-1 G > C and IVS5-1 G > A, constitute 83% of alleles.

Fatty acid synthesis from [2-14C]acetate in normal and peroxisome-deficient (Zellweger) fibroblasts.

The incorporation of [2-14C]acetate into the lipids of normal and peroxisome-deficient (Zellweger's syndrome) skin fibroblasts was examined. Most of the label was incorporated into triacylglycerol fatty acids in normal as well as Zellweger's syndrome cells. Triacylglycerols and cholesteryl esters in Zellweger's syndrome cells contained increased levels of labelled saturated and monounsaturated very long-chain fatty acids (VLCFA, that is fatty acids with more than 22 carbon atoms), in particular hexacosanoic (26:0) and hexacosaenoic (26:1) acids. As traces of labelled VLCFA with up to 32 carbon atoms were detected in triacylglycerols even in control cells it is probable that these fatty acids are formed naturally during the elongation process. Our data suggest that peroxisomes are involved in the chain shortening of the saturated and monounsaturated VLCFA.

Metabolism of trideuterated iso-lignoceric acid in rats in vivo and in human fibroblasts in culture.

Saturated very long chain fatty acids (fatty acids with greater than 22 carbon atoms; VLCFA) accumulate in peroxisomal disorders, but there is little information on their turnover in patients. To determine the suitability of using stable isotope-labeled VLCFA in patients with these disorders, the metabolism of 22-methyl[23,23,23-2H3]tricosanoic (iso-lignoceric) acid was studied in rats in vivo and in human skin fibroblasts in culture. The deuterated iso-VLCFA was degraded to the corresponding 16- and 18-carbon iso-fatty acids by rats in vivo and by normal human skin fibroblasts in culture, but there was little or no degradation in peroxisome-deficient (Zellweger's syndrome) fibroblasts, indicating that its oxidation was peroxisomal. Neither the 14-, 20-, and 22-carbon iso-fatty acids nor the corresponding odd-chain metabolites could be detected. In the rat, the organ containing most of the iso-lignoceric acid, and its breakdown products, was the liver, whereas negligible amounts were detected in the brain, suggesting that little of the fatty acid crossed the blood-brain barrier. Our data indicate that VLCFA labeled with deuterium at the omega-position of the carbon chain are suitable derivatives for the in vivo investigation of patients with defects in peroxisomal beta-oxidation because they are metabolized by the same pathways as the corresponding n-VLCFA. Moreover, as iso-VLCFA and their beta-oxidation products are readily separated from the corresponding n-fatty acids by normal chromatographic procedures, the turnover of VLCFA can be more precisely measured.

Very long-chain fatty acids in peroxisomal disease.

Fatty acids with from 24 to 28 carbon atoms (very long-chain fatty acids, VLCFA) are present in small amounts in all mammalian tissues. Even longer chain fatty acids with from 30 to 38 carbon atoms (ultra-long-chain fatty acids, ULCFA) are found in certain specialized tissues including retina, brain, and spermatozoa. In patients with inherited defects in peroxisomal structure and/or function, there is an accumulation of VLCFA in most tissues, while VLCFA and ULCFA levels are increased in brain. The most pronounced changes occur in those patients who have defects in peroxisomal assembly (Zellweger syndrome, infantile Refsum's disease, and neonatal adrenoleukodystrophy). In the brain of these individuals, ULCFA are distributed largely in molecular species of phosphatidylcholine with penta-, hexa-, and heptaenoic acids. In contrast, patients with X-linked adrenoleukodystrophy have increased levels of phosphatidylcholine with monoenoic rather than polyenoic ULCFA. A defect in a peroxisomal VLCFA CoA synthetase or ligase has been reported for these patients, but assembly of their peroxisomes is apparently normal. We speculate that ULCFA are normal products of carbon chain elongation. We have confirmed this by demonstrating the elongation of [1-14C]hexacosatetraenoic acid (26:4n-6) by rat brain in vivo to a series of longer chain tetraenoic acids with carbon chain lengths up to 34. Elongation to ULCFA can occur as well in non-neural tissues as shown by detection of labeled saturated and monoenoic fatty acids with up to 32 carbon atoms after incubation of normal and Zellweger syndrome fibroblasts with [2-14C] acetate. Increased labeling of VLCFA and ULCFA is observed in cells from patients with peroxisomal disorders. Our data suggest that ULCFA with up to at least 32 carbon atoms are formed normally, as a part of the elongation process in most mammalian tissues, and that control of carbon chain elongation is a major function of peroxisomes. Impairment of this function as occurs in peroxisomal disease results in the accumulation of VLCFA and ULCFA. The relative enrichment in normal tissues of ULCFA such as 32:6n-3 in ram and bull spermatozoa and 36:4n-6 in human and rat brain suggests a probable physiological role for this class of fatty acids in these tissues.

Economics of surgery and medical treatment in coronary artery disease.

Coronary disease is a major cause of increase in health-care costs. The disease is widespread, crippling, affects the money-making age groups and is expensive to treat whether medically or surgically. As benefits of surgery become more clearly realized, and receive a stronger statistical backing, it may be found that an expensive operation is more cost-beneficial than a series of less expensive hospital admissions. Whatever the cost-benefit or cost-effective ratios prove to be, it is likely that most patients faced with a choice between painful or pain-free life will choose the pain-free option, especially if 'the insurance will pay for it'.

Is there a ten-year valve?

Since the start of the era of exclusive use of valve replacement in 1960, the only valve that has received continuous use [since 1962] has been the aortic homograft. Results with this valve indicate about a 60- to 70-percent 10-year survival rate. The next most continuously used designs have been the Smeloff-Cutter and the 1200-1260 series Starr-Edwards ball valves. Both of these valves have survival rates after 8 years of about 70%, and are, therefore, comparable to the homografts. More recently started series of homografts valves, tissue valves and prosthetic valves of various types appear to confer a better chance of 10-year survival than older valves in older series. Not all the improvements in survival rates can be ascribed to changes in valve design. Better selection of patients, improved intraoperative myocardial protection and better postoperative care all contribute to what appears to be progressive improvement in surgical results.