Flux analysis of inborn errors of metabolism.

Patients with an inborn error of metabolism (IEM) are deficient of an enzyme involved in metabolism, and as a consequence metabolism reprograms itself to reach a new steady state. This new steady state underlies the clinical phenotype associated with the deficiency. Hence, we need to know the flux of metabolites through the different metabolic pathways in this new steady state of the reprogrammed metabolism. Stable isotope technology is best suited to study this. In this review the progress made in characterizing the altered metabolism will be presented. Studies done in patients to estimate the residual flux through the metabolic pathway affected by enzyme deficiencies will be discussed. After this, studies done in model systems will be reviewed. The focus will be on glycogen storage disease type I, medium-chain acyl-CoA dehydrogenase deficiency, propionic and methylmalonic aciduria, urea cycle defects, phenylketonuria, and combined D,L-2-hydroxyglutaric aciduria. Finally, new developments are discussed, which allow the tracing of metabolic reprogramming in IEM on a genome-wide scale. In conclusion, the outlook for flux analysis of metabolic derangement in IEMs looks promising.

From genome to phenome-Simple inborn errors of metabolism as complex traits.

Sporadically, patients with a proven defect in either mFAO or OXPHOS are described presenting with a metabolic profile and clinical phenotype expressing concurrent defects in both pathways. Biochemical linkages between both processes are tight. Therefore, it is striking that concurrent dysfunction of both systems occurs so infrequent. In this review, the linkages between OXPHOS and mFAO and the hypothesized processes responsible for concurrent problems in both systems are reviewed, both from the point of view of primary biochemical connections and secondary cellular responses, i.e. signaling pathways constituting nutrient-sensing networks. We propose that affected signaling pathways may play an important role in the phenomenon of concurrent defects. Recent data indicate that interference in the affected signaling pathways may resolve the pathological phenotype even though the primary enzyme deficiency persists. This offers new (unexpected) prospects for treatment of these inborn errors of metabolism. This article is part of a Special Issue entitled: From Genome to Function.

Pathogenesis of cognitive dysfunction in phenylketonuria: review of hypotheses.

In untreated phenylketonuria (PKU), deficiency of phenylalanine hydroxylase (PAH) results in elevated blood phenylalanine (Phe) concentrations and severe mental retardation. Current dietary treatment prevents mental retardation, but cognitive outcome remains suboptimal. The mechanisms by which elevated blood Phe concentrations disturb cerebral metabolism and cognitive function have not been fully elucidated. In this review, we discuss different hypotheses on the pathogenesis of PKU, focusing on the effects of disturbed large neutral amino acid (LNAA) transport from blood to brain on cerebral neurotransmitter and protein synthesis. Although the definitive roles of these processes in PKU pathogenesis are not fully understood yet, both substantially influence clinical outcome.

Soraphen, an inhibitor of the acetyl-CoA carboxylase system, improves peripheral insulin sensitivity in mice fed a high-fat diet.

AIM: Inhibition of the acetyl-CoA carboxylase (ACC) system, consisting of the isozymes ACC1 and ACC2, may be beneficial for treatment of insulin resistance and/or obesity by interfering with de novo lipogenesis and beta-oxidation. We have evaluated effects of pharmacological inhibition of ACC by soraphen (SP) on high fat (HF) diet-induced insulin resistance in mice. METHOD: Male C57Bl6/J mice were fed control chow, a HF diet or a HF diet supplemented with SP (50 or 100 mg/kg/day). RESULTS: Body weight gain and total body fat content of SP-treated animals were significantly reduced compared with HF-fed mice. Fractional synthesis of palmitate was significantly reduced in mice treated with SP, indicative for ACC1 inhibition. Plasma beta-hydroxybutyrate levels were significantly elevated by SP, reflecting simultaneous inhibition of ACC2 activity. Mice treated with SP showed improved peripheral insulin sensitivity, as assessed by hyperinsulinaemic euglycaemic clamps. CONCLUSION: Pharmacological inhibition of the ACC system is of potential use for treatment of key components of the metabolic syndrome.

Adiponectin levels correlate with the severity of hypertriglyceridaemia in glycogen storage disease Ia.

Glycogen storage disease type Ia (GSD Ia) is characterized by severe hypercholesterolaemia and hypertriglyceridaemia. Little is known about the aetiology of the hyperlipidaemia in GSD Ia. Adipokines play an important regulatory role in lipid metabolism. We investigated whether adipokine concentrations were correlated with the degree of hyperlipidaemia in GSD Ia patients. Six patients with GSD Ia were studied in semi-fasted conditions. Adiponectin, but not leptin, correlated (r(2) = -0.79, p = 0.02) with plasma triglyceride concentrations in the GSD Ia patients. Leptin correlated well with BMI (r(2) = 0.59, p < 0.01). However, neither body mass index (BMI) nor homeostasis model assessment (HOMA), as a marker of insulin sensitivity, correlated with triglyceride concentrations. Although a small number of patients were studied, these results indicate that adiponectin concentrations are correlated with the degree of hypertriglyceridaemia in GSD Ia. Pharmacological treatment aimed at increasing adiponectin levels might improve the metabolic status of these patients.

Neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in The Netherlands: the importance of enzyme analysis to ascertain true MCAD deficiency.

The outcome was determined of population-wide neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using tandem mass spectrometry (MS/MS) in The Netherlands, between October 2003 and September 2005. Prospective population-wide neonatal screening for MCAD deficiency was performed in the northern part of The Netherlands. In newborns with blood octanoylcarnitine (C(8:0)) concentrations > or =0.3 micromol/L, clinical and laboratory follow-up was initiated, including MCAD enzymatic measurements which played a decisive role. In a 2-year period, 66 216 newborns were investigated for MCAD deficiency and follow-up was initiated in 28 newborns. True-positives (n = 14) were identified based upon MCAD enzyme activity <50%, measured with hexanoyl-CoA as substrate. The observed prevalence of MCAD deficiency was 1/6600 (95% CI: 1/4100-1/17 400). In addition to an elevated C(8:0) concentration, a C(8:0)/C(10:0) molar ratio >5.0 turned out to differentiate between false-positives and true-positives. Measurement of MCAD activity using phenylpropionyl-CoA as a substrate further discriminated between newborns with MCAD deficiency and so-called mild MCAD deficiency. To summarize, neonatal screening for MCAD deficiency in the northern part of The Netherlands resulted in the predicted number of affected newborns. Measurement of MCAD activity in leukocytes or lymphocytes using phenylpropionyl-CoA as a substrate can be regarded as the gold standard to diagnose MCAD deficiency upon initial positive screening test results.

Phenylketonuria. The in vivo hydroxylation rate of phenylalanine into tyrosine is decreased.

In phenylketonuria (PKU), the enzyme phenylalanine hydroxylase is deficient, resulting in a decreased conversion of phenylalanine (Phe) into tyrosine (Tyr). The severity of the disease is expressed as the tolerance for Phe at 5 yr of age. In PKU patients it is assumed that the decreased conversion of Phe into Tyr is directly correlated with the tolerance for Phe. We investigated this correlation by an in vivo stable isotope study. The in vivo residual hydroxylation was quantitated using a primed continuous infusion of L-[ring- 2H5]Phe and L-[1-13C]Tyr and the determination of the isotopic enrichments of L-[ring-2H5]Phe, L-[ring-2H4]Tyr, and L-[1-13C]Tyr in plasma. Previous reports by Thompson and coworkers (Thompson, G.N., and D. Halliday. 1990. J. Clin. Invest. 86:317-322; Thompson, G.N., J.H. Walter, J.V. Leonard, and D. Halliday. 1990. Metabolism. 39:799-807; Treacy, E., J.J. Pitt, K. Seller, G.N. Thompson, S. Ramus, and R.G.H. Cotton. 1996. J. Inherited Metab. Dis. 19:595- 602), applying the same technique, showed normal in vivo hydroxylation rates of Phe in almost all PKU patients. Therefore, our study was divided up in two parts. First, the method was re-evaluated. Second, the correlation between the in vivo hydroxylation of Phe and the tolerance for Phe was tested in seven classical PKU patients. Very low (0.13- 0.95 micromol/kg per hour) and normal (4.11 and 6.33 micromol/kg per hour) conversion rates were found in patients and controls, respectively. Performing the infusion study twice in the same patient and wash-out studies of the labels at the end of the experiment in a patient and control showed that the method is applicable in PKU patients and gives consistent data. No significant correlation was observed between the in vivo hydroxylation rates and the tolerances. The results of this study, therefore, showed that within the group of patients with classical PKU, the tolerance does not depend on the in vivo hydroxylation.

Pyridoxine induces non-specific EEG alterations in infants with therapy resistant seizures.

PURPOSE: In infants with frequent therapy resistant seizures (TRS-infants), clinical detection of pyridoxine-dependency (PD) or -responsiveness (PR) occurs by empirical intravenous (IV) pyridoxine administration during recording of the EEG. However, in undiagnosed TRS-infants it is still unclear to what extent EEG alterations by pyridoxine-IV are attributable to PD/PR or to non-specific responses. Before EEG alterations by pyridoxine-IV can be ascribed to PD/PR, these non-specific responses should be excluded first. METHODS: In 10 TRS-infants under 1 year of age, we determined the EEG effect by pyridoxine-IV on the EEG-recording. RESULTS: After pyridoxine-IV administration, our data indicate declined (10-15%; p<0.05) EEG-amplitudes and total power (magnitude/frequency-band) at frontal, central and centro-temporal electrodes. CONCLUSION: In TRS-infants, pyridoxine-IV affects EEG-amplitude and -total power in a non-specific way, which does not identify PD/PR.

Evidence against a MgATP-dependent proton pump in rat-liver lysosomes.

1. The effect of MgATP has been studied on the accumulation of the lipid-soluble anion thiocyanate, the accumulation of the lipid-soluble base methylamine, and the fluorescence of bound anilinonaphthalene sulphonate in rat-liver lysosomes. The lysosomes used were isolated from the livers of rats pretreated with Triton WR 1339. 2. The accumulation of thiocyanate is stimulated by the addition of valinomycin in the presence of K+ but not by the addition of MgATP. 3. The fluorescence of anilinonaphthalene sulphonate bound to lysosomes is enhanced by valinomycin in the presence of K+, the extent of the enhancement being dependent on the concentration of K+. In contrast, MgATP has no effect on the fluorescence. 4. The intralysosomal pH, as estimated from the distribution of methylamine, is not affected by the addition of MgATP in media with or without K+, Na+ or HCO3-. 5. These data strongly suggest that there is no MgATP-dependent proton pump in rat-liver lysosomes.

Effect of ionophores on intralysosomal pH.

1. The effect of ionophores on the intralysosomal pH (as estimated from the distribution of a weak acid or base), on the distribution of 42K+ across the lysosomal membrane, and on the intralysosomal degradation of 125I-labelled bovine serum albumin has been studied. 2. Nigericin and X537A equilibrate both 42K+ and H+ across the lysosomal membrane. Gramicidin equilibrates H+ across the lysosomal membrane, this equilibration being more effective in a NaCl than in a KCl medium. Thus all three ionophores exhibit the same ion specificity as in other membranes. 3. The effect of the exchange-diffusion ionophores cannot be imitated by the combination of valinomycin with an uncoupler. Valinomycin by itself also has no effect. 4. X537A and nigericin inhibit the intralysosomal degradation of 125I-labelled albumin only when potassium is present. In a sucrose-containing medium no effect is found. Similar results were obtained with gramicidin. 5. These data suggest that the lysosomal membrane is impermeable to monovalent cations at 25 or 37 degrees C, and that the transport of protons is organised in such a way that electroneutrality is maintained.

Relationship between medium pH and that of the lysosomal matrix as studied by two independent methods.

1. The method of estimating the intralysosomal pH by measuring the distribution of [14C]methylamine in lysosomes isolated from the livers of Triton WR 1339-treated rats has been critically examined. 2. In lysed lysosomes, methylamine is bound to the membrane fragments, but this binding can be completely suppressed by increasing the concentration of monovalent cations in the medium. 3. In intact lysosomes, the binding of [14C]methylamine is only partly inhibited by monovalent cations at 25 degrees C. 4. THe accumulation of [14C]methylamine in intact lysosomes is progressively inhibited as the concentration of methylamine is increased. A similar inhibition of [14C]methylamine accumulation is obtained with NH4Cl. 5. Similar values for the intralysosomal pH were obtained from measurements of the distribution of methylamine, dimethylamine and trimethylamine, which are accumulated in the lysosomes, and of 5,5-dimethyloxazolidinedione-2,4, which is excluded. 6. The breakdown of endocytosed 123I-labelled bovine serum albumin by intact isolated lysosomes is much less sensitive to the pH of the medium than the breakdown of added protein by lysed lysosomes. 7. The intralysosomal pH has been estimated by comparing the rate of breakdown of endocytosed 125I-labelled albumin in intact lysosomes as a function of medium pH with that of added 125I-labelled albumin by lysed lysosomes at different pH values. The values obtained agree well with those calculated from the distribution of [14C]methylamine. 8. Methylamine and NH4Cl inhibit the breakdown of 125I-labelled albumin in intact lysosomes, particularly at high medium pH, but have no effect on the breakdown by lysed lysosomes. 9. It is concluded that a pH difference across the lysosomal membrane (more acidic inside than outside) is maintained by the presence of indiffusible negatively charged groups within the lysosomes, and by the permeation across the lysosomal membrane of protons together with permeant anions (or of OH- in exchange for anions).

Acute inhibition of glucose-6-phosphate translocator activity leads to increased de novo lipogenesis and development of hepatic steatosis without affecting VLDL production in rats.

Glucose-6-phosphatase (G6Pase) is a key enzyme in hepatic glucose metabolism. Altered G6Pase activity in glycogen storage disease and diabetic states is associated with disturbances in lipid metabolism. We studied the effects of acute inhibition of G6Pase activity on hepatic lipid metabolism in nonanesthetized rats. Rats were infused with an inhibitor of the glucose-6-phosphate (G6P) translocator (S4048, 30 mg. kg(-1). h(-1)) for 8 h. Simultaneously, [1-(13)C]acetate was administered for determination of de novo lipogenesis and fractional cholesterol synthesis rates by mass isotopomer distribution analysis. In a separate group of rats, Triton WR 1339 was injected for determination of hepatic VLDL-triglyceride production. S4048 infusion significantly decreased plasma glucose (-11%) and insulin (-48%) levels and increased hepatic G6P (201%) and glycogen (182%) contents. Hepatic triglyceride contents increased from 5.8 +/- 1.4 micromol/g liver in controls to 20.6 +/- 5.5 micromol/g liver in S4048-treated animals. De novo lipogenesis was increased >10-fold in S4048-treated rats, without changes in cholesterol synthesis rates. Hepatic mRNA levels of acetyl-CoA carboxylase and fatty acid synthase were markedly induced. Plasma triglyceride levels increased fourfold, but no differences in plasma cholesterol levels were seen. Surprisingly, hepatic VLDL-triglyceride secretion was not increased in S4048-treated rats. These studies demonstrate that inhibition of the G6Pase system leads to acute stimulation of fat synthesis and development of hepatic steatosis, without affecting hepatic cholesterol synthesis and VLDL secretion. The results emphasize the strong interactions that exist between hepatic carbohydrate and fat metabolism.

Large daily fluctuations in plasma tyrosine in treated patients with phenylketonuria.

In patients with phenylketonuria (PKU), extra tyrosine supplementation is advocated in addition to tyrosine-enriched amino acid mixtures. PKU patients have low fasting plasma tyrosine concentrations, but little is known about tyrosine fluctuations during the day. Plasma tyrosine concentrations were studied in 12 PKU patients in response to a test without breakfast and to three tests with different tyrosine contents in breakfast and lunch: 0%/30%, 25%/30%, 50%/10%, and 75%/10% tests, reflecting the protein consumption at breakfast and lunch, respectively. Prolonged fasting resulted in a small decrease in the already low overnight fasting plasma tyrosine concentrations. Breakfast and lunch with 25% and 30% of the daily tyrosine intake resulted in both lower than normal and higher than normal tyrosine concentrations. The 50%/10% and 75%/10% tests resulted in excessively high plasma tyrosine concentrations in most patients. Therefore, both lower than normal and higher than normal postprandial plasma tyrosine concentrations were found in treated PKU patients, even if the daily tyrosine intake was distributed evenly. When there was a large fractional tyrosine intake from one meal, very high plasma tyrosine concentrations were found. Therefore, strict control of plasma tyrosine is necessary if tyrosine supplementation is considered in addition to the tyrosine-enriched amino acid mixtures.

Resting oxygen consumption and in vivo ADP are increased in myopathy due to complex I deficiency.

BACKGROUND: Patients with isolated complex I deficiency (CID) in skeletal muscle mitochondria often present with exercise intolerance as their major clinical symptom. OBJECTIVE: To study the in vivo bioenergetics in patients with complex I deficiency in skeletal muscle mitochondria. METHODS: In vivo bioenergetics were studied in three of these patients by measuring oxygen uptake at rest and during maximal exercise, together with forearm ADP concentrations ([ADP]) at rest. Whole-body oxygen consumption at rest (VO(2)) was measured with respiratory calorimetry. Maximal oxygen uptake (VO(2)max) was measured during maximal exercise on a cycle ergometer. Resting [ADP] was estimated from in vivo (31)P MRS measurements of inorganic phosphate, phosphocreatine, and ATP content of forearm muscle. RESULTS: Resting VO(2) was significantly increased in all three patients: 128 +/- 14% (SD) of values in healthy control subjects. VO(2)max in patients was on average 2.8 times their VO(2) at rest and was only 28% of VO(2)max in control subjects. Resting [ADP] in forearm muscle was significantly increased compared with healthy control subjects (patients 26 +/- 2 microM, healthy controls 9 +/- 2 microM). CONCLUSION: In patients with CID, the increased whole-body oxygen consumption rate at rest reflects increased electron transport through the respiratory chain, driven by a decreased phosphorylation potential. The increased electron transport rate may compensate for the decreased efficiency of oxidative phosphorylation (phosphorylation potential).

Phenylketonuria: plasma phenylalanine responses to different distributions of the daily phenylalanine allowance over the day.

OBJECTIVE: To achieve smooth control of plasma phenylalanine concentrations in phenylketonuric patients, it is advocated to divide the daily intake of natural protein and amino acid supplements equally over the meals. However, this may be quite an encumbrance for the patient. We, therefore, investigated whether a breakfast with an unequal daily distribution results in an undue rise in the plasma phenylalanine concentration. DESIGN: Plasma phenylalanine concentrations were measured in seven patients with phenylketonuria in response to three tests with breakfast and lunch, representing an equally or unequally divided daily distribution of the individually tailored phenylalanine intake. Breakfast contained 25%, 50%, or 75%, whereas lunch contained 30% or 10% of the individual daily phenylalanine allowance, respectively. RESULTS: Plasma phenylalanine concentrations showed postprandial increases of up to 26% above baseline. Generally, phenylalanine returned to baseline during the test and remained within the target range if baseline phenylalanine was within that range. Two patients having values in the upper target range showed a rise just above the target range for 60 minutes on an unequal daily distribution of phenylalanine. In another patient treated similarly, plasma phenylalanine did not return to baseline during the test. CONCLUSIONS: Unequal distributions of the daily phenylalanine allowance are justified, provided that the patient is in good clinical condition, adjusted to the diet adequately, and the daily allowance is not exceeded. At this time, however, we cannot recommend this unequal daily distribution for daily practice.

Plasma phenylalanine and tyrosine responses to different nutritional conditions (fasting/postprandial) in patients with phenylketonuria: effect of sample timing.

OBJECTIVE: To evaluate the adequacy of dietary treatment in patients with phenylketonuria, the monitoring of plasma phenylalanine and tyrosine concentrations is of great importance. The preferable time of blood sampling in relation to the nutritional condition during the day, however, is not known. It was the aim of this study to define guidelines for the timing of blood sampling with a minimal burden for the patient. DESIGN: Plasma concentrations of phenylalanine and tyrosine were measured in nine patients with phenylketonuria who had no clinical evidence of tyrosine deficiency. These values were measured during the day both after a prolonged overnight fast, and before and after breakfast. RESULTS: Phenylalanine showed a small rise during prolonged fasting, while tyrosine decreased slightly. After an individually tailored breakfast, phenylalanine remained stable, while tyrosine showed large fluctuations. CONCLUSION: It is concluded that the patient's nutritional condition (fasting/postprandial) is not important in the evaluation of the phenylalanine intake. To detect a possible tyrosine deficiency, however, a single blood sample is not sufficient and a combination of a preprandial and postprandial blood sample on the same day is advocated.

Determination of low isotopic enrichment of L-[1-13C]valine by gas chromatography/combustion/isotope ratio mass spectrometry: a robust method for measuring protein fractional synthetic rates in vivo.

A method was developed for measuring protein fractional synthetic rates using the N-methoxycarbonylmethyl ester (MCM) derivative of L-[1-13C]valine and on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The derivatization procedure can be performed rapidly and GC separation of valine from the other branched-chain amino acids, leucine and isoleucine, is easily obtained. A good linear relationship was observed between the increment of the 13C/12C isotope ratio in CO2 gas derived from the combustion of derivatized valine and the tracer mole ratio of L-[1-13C]valine to unlabelled valine. The limit of quantitation was at an L-[1-13C]valine tracer mole ratio of 0.0002. The method was used to measure the isotopic enrichment of L-[1-13C]valine in standard mixtures and in skeletal muscle of six growing piglets infused with L-[1-13C]valine (2 mg kg-1 h-1 for 6 h). After infusion of L-[1-13C]valine the mean tracer mole ratio in plasma of L-[1-13C]valine at the isotopic steady state was 0.0740 +/- 0.0056 (GC/MS, mean +/- SEM) and the mean tracer mole ratio of valine in muscle protein fraction at 6 h was 0.000236 +/- 0.000038 (GC/C/IRMS). The resulting mean protein fractional synthetic rate in piglet skeletal muscle was 0.052 +/- 0.007% h-1, which is in good agreement with literature data obtained with alternative, more elaborate techniques. By this method protein fractional synthetic rates can be measured at low isotopic enrichment levels using L-[1-13C]valine, the MCM derivative and on-line GC/C/IRMS.

Stable isotope dilution analysis of cystine in granulocyte suspensions as cysteine: a powerful method for the diagnosis, the follow-up, and treatment of patients with cystinosis.

A stable isotope dilution method was developed for the determination of cystine in granulocytes. Granulocytes were isolated from blood samples of treated cystinosis patients. Cystine in the granulocyte suspension was decoupled from proteins and converted to cysteine by treatment with a tri-butyl phosphine solution. Tertiary butyldimethyl silyl derivatives were prepared and analyzed by gas chromatography/mass spectrometry. Selective ion monitoring was carried out at m/z 304.3 (M-159 and m/z 406.4 (M-57) for the natural, and at m/z 306.3 and 408.4 for the labelled compound. [3,3,3',3'-2H]-DL-cystine was used as internal standard for the isotope dilution analysis. Concentrations of cystine in granulocytes could be accurately measured. There was a distinct difference in cystine concentrations in healthy individuals and treated patients.

Determination of medium chain acyl-CoA dehydrogenase activity in cultured skin fibroblasts using mass spectrometry.

Medium chain acyl-CoA dehydrogenase deficiency, a defect of mitochondrial beta-oxidation, is one of the most frequently occurring among inborn errors of metabolism. We describe a rapid and sensitive gas chromatographic/mass spectrometric method allowing reliable assessment of medium chain acyl-CoA dehydrogenase activity in cultured skin fibroblasts. We investigated MCAD activity in three presumed medium chain acyl-CoA dehydrogenase deficient (MCADD) patients and 10 control subjects. The medium chain acyl-CoA dehydrogenase activity determined in three patients was 1.0 +/- 0.4 nmol.min-1.mg-1 protein (mean +/- SD; range: 0.6-1.4) and in controls it was 2.8 +/- 1.0 nmol.min-1.mg-1 protein (mean +/- SD; range: 1.6-4.4).

Low diagnostic value of fasting and post-methionine load homocysteine tests. A study in Dutch subjects with homocysteine test indications.

BACKGROUND: Homocysteine is a cardiovascular disease risk factor. We investigated, both in subjects with past plasma total homocysteine (tHcy) test indications and healthy adults, the diagnostic value of a fasting (tHcy) (f-tHcy) and the added value of a post-methionine-load tHcy (postload-tHcy). METHODS: Plasma homocysteine cut-off values were retrospectively used for hyperhomocysteinemia assessment in 3477 subjects with past tHcy test indications and 177 apparently healthy subjects. Cut-off values were based on reference limits (f-tHcy < or = 15.0; postload-tHcy < or = 50.0 micro mol/l), relative risk (f-tHcy < or = 12.0, postload-tHcy < or = 38.0; or f-tHcy < or = 10.0 micro mol/l) and vitamin-optimized reference limits (f-tHcy < or = 9.3; postload-tHcy < or = 35.1 micro mol/l). RESULTS: Use of the American Heart Association 10 micro mol/l f-tHcy cut-off value gave hyperhomocysteinemia prevalences of 65% in subjects with past tHcy test indications and 50% in healthy subjects. The combination of the vitamin-optimized reference limits for f-tHcy and postload-tHcy gave a hyperhomocysteinemia prevalence of 79% in subjects with tHcy test indications, of which only 5% was on account of increased postload-tHcy. Corresponding values for healthy subjects were 68% and 3%, respectively. CONCLUSIONS: Employment of a 10 micro mol/l (American Heart Association) or 9.3 micro mol/l (vitamin-optimized reference) cut-off value leaves no indications for tHcy testing from an evidence-based point-of-view.