Refsum disease is an autosomal-recessively inherited disorder characterized clinically by a tetrad of abnormalities: retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia and elevated protein levels in the cerebrospinal fluid (CSF) without an increase in the number of cells in the CSF. All patients exhibit accumulation of an unusual branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), in blood and tissues. Biochemically, the disease is caused by the deficiency of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal protein catalyzing the first step in the alpha-oxidation of phytanic acid. We have purified PhyH from rat-liver peroxisomes and determined the N-terminal amino-acid sequence, as well as an additional internal amino-acid sequence obtained after Lys-C digestion of the purified protein. A search of the EST database with these partial amino-acid sequences led to the identification of the full-length human cDNA sequence encoding PhyH: the open reading frame encodes a 41.2-kD protein of 338 amino acids, which contains a cleavable peroxisomal targeting signal type 2 (PTS2). Sequence analysis of PHYH fibroblast cDNA from five patients with Refsum disease revealed distinct mutations, including a one-nucleotide deletion, a 111-nucleotide deletion and a point mutation. This analysis confirms our finding that Refsum disease is caused by a deficiency of PhyH.

Adult↗

PubMed1997

Does metformin increase the serum total homocysteine level in non-insulin-dependent diabetes mellitus?

OBJECTIVE: The aim of this study was to estimate the effect of metformin on the serum total homocysteine level in non-insulin-dependent diabetes mellitus (NIDDM) patients. An elevated serum total homocysteine level is a risk factor for atherosclerosis. Metformin decreases serum vitamin B12, and may thereby indirectly increase the serum total homocysteine level. DESIGN: A cross-sectional study in a primary care setting. SUBJECTS, MAIN OUTCOME MEASURES: Fasting serum total homocysteine level was measured in 40 NIDDM patients who had received treatment with metformin (500-2550 mg per day) for at least six months, and in 71 NIDDM patients not treated with metformin and matched for sex, age (+/- 5 years), serum creatinine (+/- 5 micromol L[-1]) and current smoking habits. 'Exposed' patients were matched with 'nonexposed' patients. A two-way analysis of variance was performed. RESULTS: The mean serum total homocysteine level was 11.5 micromol L(-1) in the metformin-exposed patients and 10.6 micromol L(-1) in the nonexposed patients. Thus, the metformin-exposed patients had slightly higher serum total homocysteine levels (difference 0.8 micromol L(-1), 95% confidence interval (-0.4-2.0 micromol L[-1]). Results were similar in men and women. Finally, no dose-response relationship between cumulative exposure to metformin (dose x duration of treatment) and the serum total homocysteine level could be demonstrated. CONCLUSION: We conclude that the effect of metformin on serum total homocysteine level in NIDDM patients, if any, is likely to be small.

Adult↗

PubMed1997

L-2-hydroxyglutaric aciduria: clinical heterogeneity versus biochemical homogeneity in a sibship.

Three out of four sibs in a North-African family were affected with L-2-hydroxyglutaric aciduria. The youngest sib was most severely handicapped: she was diagnosed at 2.5 years of age, whereas the then 7- and 10-year-old siblings had a less pronounced psychomotor retardation. All patients had an increased head circumference in contrast to the healthy, non-affected sibling. Urine and plasma levels of L-2-hydroxyglutaric acid in the three sibs were similar and showed only a small variation. Magnetic resonance imaging (MRI) of the brain in the eldest sib showed hyperintense signal on T2-weighted images of the basal ganglia, dentate nucleus and subcortical white matter. The youngest sib showed identical white matter abnormalities of the corpus medullare cerebelli. These abnormalities were consistent with demyelination and/or spongiosis. On two occasions cerebrospinal fluid amino acid chromatography in the youngest sib showed an increased concentration of lysine and a decreased level of glutamine. Plasma lysine was normal. It is concluded that L-2-hydroxyglutaric aciduria is almost invariably associated with neurological disease; the severity of the symptoms does not seem to be completely dependent on the extent of the biochemical abnormalities and may even be variable within a family.

Ataxia↗

PubMed1997

Hyperhomocysteinaemia and protein S deficiency in complicated pregnancies.

OBJECTIVE: The aim of our study was to investigate whether women with placental abruption, intrauterine fetal death or small for gestational age infants have metabolic and/or haemostatic abnormalities which are known to be risk factors for intravascular thrombosis. DESIGN: For two years blood tests were performed at > 10 weeks after delivery on all women without hypertensive disorders either before or during pregnancy, who had been consecutively admitted to our hospital with placental abruption, intrauterine fetal death and small for gestational age. SAMPLE: A total of 62 women who had placental abruption (n = 31), intrauterine fetal death (n = 18) and a small for gestational age infant (n = 13). SETTING: Obstetric outpatient clinic in a university hospital (Free University Hospital, Amsterdam). METHODS: Presence of hyperhomocysteinaemia, various coagulation abnormalities and anticardiolipins was investigated. RESULTS: Abnormalities were found in 20 women in the placental abruption group (20/31, 65%), in 10 women in the intrauterine fetal death group (10/18, 56%) and in 11 women in the small for gestational age group (11/13, 85%). Eight out of these 31 women had more than one abnormality. In the group of 62 women protein S deficiency was demonstrated in 26%, hyperhomocysteinaemia in 24%, Protein C deficiency in 6%, anticardiolipin IgG in 11%, anticardiolipin IgM in 5%, Lupus anticoagulant in 2%. An antithrombin III deficiency was not found. Thirty-three women were tested for activated protein C resistance (9% positive) and factor V Leiden mutation (6% positive). Hyperhomocysteinaemia was treated with a daily oral dose of 250 mg pyridoxine and 5 mg folic acid. After six weeks of vitamin supplementation homocysteine levels were tested again. At that time a mean reduction of fasting homocysteine value of 68% (95% CI 57-79) was found and of post-load value of 65% (95% CI 55-76). CONCLUSIONS: Based on the results of our study, it can be concluded that women whose pregnancies are complicated by either placental abruption, intrauterine fetal death or small for gestational age, even if there is no history of thrombo-embolic disorders or hypertension during pregnancy, should be advised to undergo an examination for metabolic and/or haemostatic abnormalities.

Abruptio Placentae↗

PubMed1997

The clinical phenotype of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria): case reports of 23 new patients.

OBJECTIVES: To further define the clinical spectrum of the disease for pediatric and metabolic specialists, and to suggest that the general pediatrician and pediatric neurologist consider succinic semialdehyde dehydrogenase (SSADH) deficiency in the differential diagnosis of patients with (idiopathic) mental retardation and emphasize the need for accurate, quantitative organic acid analysis in such patients. PATIENTS: The clinical features of 23 patients (20 families) with SSADH deficiency (4-hydroxybutyric acid-uria) are presented. The age at diagnosis ranged from 3 months to 25 years in the 11 male and 12 female patients; consanguinity was noted in 39% of families. OUTCOME MEASUREMENTS: The following abnormalities were observed (frequency in 23 patients): motor delay, including fine-motor skills, 78%; language delay, 78%; hypotonia, 74%; mental delay, 74%; seizures, 48%; decreased or absent reflexes, 39%; ataxia, 30%; behavioral problems, 30%; hyperkinesis, 30%; neonatal problems, 26%; and electroencephalographic abnormalities, 26%. Associated findings included psychoses, cranial magnetic resonance or computed tomographic abnormalities, and ocular problems in 22% or less of patients. Therapy with vigabatrin proved beneficial to varying degrees in 35% of the patients. Normal early development was noted in 30% of patients. CONCLUSIONS: Our data imply that two groups of patients with SSADH deficiency exist, differentiated by the course of early development. Our recommendation would be that accurate, quantitative organic acid analysis in an appropriate specialist laboratory be requested for any patients presenting with two or more features of mental, motor, or language delay and hypotonia of unknown cause. Such analyses are the only definitive way to diagnose SSADH deficiency; the diagnosis can be confirmed by determination of enzyme activity in white cells from whole blood. We think that increased use of organic acid determination will lead to increased diagnosis of SSADH deficiency and a more accurate representation of disease frequency. As additional patients are identified, we should have a better understanding of both the metabolic and clinical profiles of SSADH deficiency.

Adolescent↗

PubMed1997

Quantitative analysis of plasma acylcarnitines using gas chromatography chemical ionization mass fragmentography.

A stable isotope dilution gas chromatography chemical ionization mass spectrometry (GC-CI-MS) method was developed for the quantitative profiling of plasma acylcarnitines. The clean-up procedure was comprised of a solid-phase cation exchange extraction using PRS-columns from which the acylcarnitines were eluted with a barium chloride solution. Isolated acylcarnitines were transformed into acyloxylactones and analyzed by positive GC-CI-MS using isobutane as reactant gas. The selected monitoring of a common ion at m/z [85]+ and the protonated molecular ion enabled a selective and sensitive detection of all C2-C18 acylcarnitines. An accurate quantitation was achieved by the use of stable isotope-labeled internal standards (C2-C18) and acylcarnitines could be analyzed in the sub-nanomolar range. Control values for C2-C18 acylcarnitines in plasma were established. Concentrations ranged from 0.02 micromol/L for C14-acylcarnitine to 4.90 micromol/L for C2-acylcarnitine. The diagnostic suitability of the method was demonstrated for patients with medium-chain acyl-CoA dehydrogenase deficiency and very long-chain acyl-CoA dehydrogenase deficiency.

Acyl-CoA Dehydrogenase↗

PubMed1997

In vivo stable isotope studies in three patients affected with mitochondrial fatty acid oxidation disorders: limited diagnostic use of 1-13C fatty acid breath test using bolus technique.

The in vivo oxidation of fatty acids (FA) of different chain length was investigated in three patients with documented mitochondrial FA oxidation disorders: one patient with mild multiple acyl-CoA dehydrogenase deficiency (MADM), one with medium chain acyl-CoA dehydrogenase deficiency (MCAD), and one with carnitine palmitoyltransferase I deficiency (CPT I). Breath tests were performed after oral administration of 1-13C butyric. 1-13C octanoic, and 1-13C palmitic acids. 13C/12C ratio in the expired oxidative end product CO2 was measured. The cumulative 13C elimination was calculated and expressed as a percentage of the administered dose. In the MADM patient the influence of carnitine therapy (or deprivation) on the utilization of 1-13C palmitic acid was also examined. In the MCAD and CPT I patients, the 1-13C butyric, 1-13C octanoic and 1-13C palmitic acids in vivo oxidation were similar to five healthy controls. In the MADM patient, the oxidation of 1-13C butyric and 1-13C octanoic acids were normal, whereas the metabolism of 1-13C palmitic acid ranged from 33% of 66% of controls. In this patient the serum carnitine level decreased from 60 to 27 mumol/l without carnitine supplementation. Clinically there was mild hypotonia. 1-13C palmitic acid oxidation compared to controls was 50%. After 2 further weeks of carnitine deprivation the serum carnitine was 10-15 mumol/l. Clinically he was very hypotonic and had a large liver. 1-13C Palmitic acid oxidation was 33%. After 6 weeks of readministration of carnitine (L-carnitine 100 mg/kg/day p.o.) the serum carnitine was 60 mumol/l and the patient was in good clinical condition. 1-13C palmitic acid oxidation was 66% compared to controls. Our study implies that this simple fatty acid breath test is not of diagnostic use for detection of enzymatic defects in FA oxidation disorders. The carnitine dependent 1-13C palmitic acid oxidation indicates that this test might be of some value in cases with primary or secondary carnitine deficiencies.

Acyl-CoA Dehydrogenase↗

PubMed1997

Phytanic acid alpha-oxidation: decarboxylation of 2-hydroxyphytanoyl-CoA to pristanic acid in human liver.

The degradation of the first intermediate in the alpha-oxidation of phytanic acid, 2-hydroxyphytanoyl-CoA, was investigated. Human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA or 2-hydroxyphytanic acid, after which formation of 2-ketophytanic acid and pristanic acid were studied. 2-Hydroxyphytanic acid was converted into 2-ketophytanic acid and pristanic acid. When ATP, Mg2+, and coenzyme A were added to the incubation medium, higher amounts of pristanic acid were formed, whereas the formation of 2-ketophytanic acid strongly decreased. When 2-hydroxyphytanoyl-CoA was used as substrate, there was virtually no 2-ketophytanic acid formation. However, pristanic acid was formed in higher amounts than with 2-hydroxyphytanic acid as substrate. This reaction was stimulated by NAD+ and NADP+. Pristanic acid, and not pristanoyl-CoA was found to be the product of the reaction. These results suggest the existence of two pathways for decarboxylation of 2-hydroxyphytanic acid. The first one, starting from 2-hydroxyphytanic acid, involves the formation of 2-ketophytanic acid with only a small amount of pristanic acid being formed. The second pathway, which starts from 2-hydroxyphytanoyl-CoA, does not involve 2-ketophytanic acid and generates higher amounts of pristanic acid. The first pathway, which is peroxisomally localized, was found to be deficient in Zellweger syndrome, whereas the second pathway, localized in microsomes, was normally active. We conclude that the second pathway is predominant under in vivo conditions.

Biomarkers↗

PubMed1996

Phytanoyl-CoA hydroxylase is present in human liver, located in peroxisomes, and deficient in Zellweger syndrome: direct, unequivocal evidence for the new, revised pathway of phytanic acid alpha-oxidation in humans.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which accumulates in a number of inherited diseases in human. Because beta-oxidation is blocked by the methyl group at C-3, phytanic acid first undergoes decarboxylation via an alpha-oxidation mechanism. The structure and subcellular localization of the phytanic acid alpha-oxidation pathway have remained enigmatic through the years, although they have generally been assumed to involve phytanic acid and not its CoA-ester. This view has recently been challenged by the findings that in rat liver phytanic acid first has to be activated to its CoA-ester before alpha-oxidation and by the discovery of a new enzyme, phytanoyl-CoA hydroxylase, which converts phytanoyl-CoA to 2-hydroxyphytanoyl-CoA. We now show that this newly discovered enzyme is also present in human liver. Furthermore, we show that this enzyme is located in peroxisomes and deficient in liver from Zellweger patients who lack morphologically distinguishable peroxisomes, which provides an explanation for the long-known deficient oxidation of phytanic acid in these patients. These results suggest that phytanic acid alpha-oxidation is peroxisomal and that it utilizes the coenzyme A derivative as substrate, thus giving further support in favour of the new, revised pathway of phytanic acid alpha-oxidation.

Cell Compartmentation↗

PubMed1996

Coexistence of hereditary homocystinuria and factor V Leiden--effect on thrombosis.

BACKGROUND: Venous and arterial thromboembolism occurs in only about one third of patients homozygous for homocystinuria, which suggests that other, contributory factors are necessary for the development of thrombosis in these patients. Factor V Leiden, an R506Q mutation in the gene coding for factor V, is the most common cause of familial thrombosis and could be a potentiating factor. METHODS: We determined activated partial-thromboplastin times in the presence and absence of activated protein C and tested for the factor V Leiden mutation in 45 members of seven unrelated consanguineous kindreds in which at least 1 member was homozygous for homocystinuria. RESULT: Thrombosis (venous, arterial, or both) occurred in 6 of 11 patients with homocystinuria (age, 0.2 to 8 years). All six also had the factor V Leiden mutation. One patient with prenatally diagnosed homocystinuria who was also heterozygous for factor V Leiden has received warfarin therapy since birth and has not had thrombosis (age, 18 months). Of four patients with homocystinuria who did not have factor V Leiden, none had thrombosis (ages at this writing, 1 to 17 years). Three women who were heterozygous for both homocystinuria and factor V Leiden had recurrent fetal loss and placental infarctions. CONCLUSIONS: Patients with concurrent homocystinuria and factor V Leiden can have an increased risk of thrombosis. Screening for factor V Leiden may be indicated in patient with homocystinuria and their family members.

Adolescent↗

PubMed1996

3-, 6- and 7-hydroxyoctanoic acids are metabolites of medium-chain triglycerides and excreted in urine as glucuronides.

Three new metabolites of medium-chain fatty acid oxidation, 3-, 6- and 7-hydroxyoctanoyl beta-D-glucuronide, were identified in the urine of six infants who were fed a diet enriched in medium-chain triglycerides (MCT). Glucuronides were extracted from the urine by organic solvent extraction with ethyl acetate and by solid-phase extraction on Sep-Pak C18 cartridges. The compounds of interest were also purified from the organic solvent extract by preparative one-dimensional thin-layer chromatography. Cleavage of the glucuronides was achieved by either alkaline hydrolysis or enzymatic hydrolysis with beta-D-glucuronidase. The analyses of the trimethylsilylated derivatives were performed both by gas chromatography with flame ionization detection (GC/FID) and by gas chromatography/mass spectrometry (GC/MS). The structure of the hydroxyoctanoic acids was proved by comparison of their mass spectra with those of reference substances. Authentic 6-hydroxyoctanoic acid was synthesized. The presence of 6-hydroxyoctanoyl glucuronide shows that in addition to beta-oxidation, omega-oxidation and (omega-1)-hydroxylation, medium-chain fatty acids can be oxidized at the omega-2 position. The conjugation of medium-chain hydroxy-monocarboxylic acids with glucuronic acid has not been described in humans before.

Caprylates↗

PubMed1996

A case of tyrosinaemia type I with normal level of succinylacetone in the amniotic fluid.

Prenatal diagnosis of tyrosinaemia type I can be achieved in cultured amniotic cells and in chorionic villus material by testing the activity of fumarylacetoacetate hydrolase and by DNA analysis, and in amniotic fluid by succinylacetone measurement. This specific metabolite can be measured either by gas chromatography-mass spectrometry or by delta-aminolevulinate dehydratase inhibition assay. In a series of 65 at-risk cases tested with the enzyme inhibition assay, one case out of the 18 with the disease had a normal level of succinylacetone. This case is presented.

Amino Acid Metabolism, Inborn Errors↗