Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening.

BACKGROUND: The most widely used method for newborn screening for homocystinuria (HCU) is a semi-quantitative bacterial inhibition assay for measuring methionine concentration in dried blood spots (DBS). Because this method has resulted in a number of missed cases due to many factors, we developed a high performance liquid chromatography (HPLC) method with fluorescence detection to measure total homocysteine (tHcy) in DBS which might be useful for newborn screening for HCU. METHODS: One disk of DBS 3 mm in diameter was sonicated in 10 min. The extract was reduced with dithioerythritol and was derivatized with 4-aminosulfonyl-7fluoro-2,1,3-benzoxadiazole before injection into HPLC. RESULTS: This method showed good linearity (r = 0.996), precision (coefficient of variation range 2.7-5%), and excellent correlation coefficient between DBS and serum tHcy, both in control (r = 0.932) and patient samples (r = 0.952). By this method, the mean tHcy concentration in DBS of preterm newborns, full-term newborns, and adults was 1.4 +/- 1.0, 2.5 +/- 1.6, and 4.9 +/- 1.5 micro mol/L, respectively. The mean tHcy DBS concentration in two cases of cystathionine-beta-synthase deficiency and one case of 5,10-methylentetrahydrofolate reductase deficiency was 22.7 +/- 2.88, 29.3 +/- 1.90, and 41.3 micro mol/L, respectively. CONCLUSIONS: The present method, which is rapid, user friendly and reliable, seems applicable to newborn screening of HCU in place of methionine measurement.

Oxidative stress and platelet activation in homozygous homocystinuria.

BACKGROUND: Severe hyperhomocysteinemia due to cystathionine beta-synthase deficiency (CbetaSD) is associated with early atherothrombotic vascular disease. Homocysteine may exert its effects by promoting oxidative damage. In the present study, we investigated whether in vivo formation of 8-iso-prostaglandin (PG) F(2alpha), a platelet-active product of arachidonic acid peroxidation, is enhanced in CbetaSD and whether it correlates with in vivo platelet activation, as reflected by thromboxane (TX) metabolite excretion. METHODS AND RESULTS: Urine and blood samples were obtained from patients with homozygous CbetaSD (n=13) and age-matched healthy subjects. Urinary 8-iso-PGF(2alpha) excretion was significantly higher in CbetaSD patients than in control subjects (640+/-384 versus 213+/-43 pg/mg creatinine; P=0.0015) and correlated with plasma homocysteine (rho=0.398, P=0.0076). Similarly, urinary 11-dehydro-TXB(2) excretion was enhanced in CbetaSD (1166+/-415 versus 324+/-72 pg/mg creatinine; P=0.0015) and correlated with urinary 8-iso-PGF(2alpha) (rho=0.362, P=0.0153). Vitamin E supplementation (600 mg/d for 2 weeks) was associated with a statistically significant increase in its plasma levels (from 16.6+/-4.6 to 40.4+/-8.7 micromol/L, P=0.0002) and with reductions in 8-iso-PGF(2alpha) (from 790+/-159 to 559+/-111 pg/mg creatinine, P=0.018) and 11-dehydro-TXB(2) (from 1273+/-383 to 913+/-336 pg/mg creatinine, P=0.028). A statistically significant inverse correlation was found between urinary 8-iso-PGF(2alpha) and plasma vitamin E levels (rho=-0.745, P=0.0135). CONCLUSIONS: The results of the present study suggest that enhanced peroxidation of arachidonic acid to form bioactive F(2)-isoprostanes may represent an important mechanism linking hyperhomocysteinemia and platelet activation in CbetaSD patients. Moreover, they provide a rationale for dose-finding studies of vitamin E supplementation in this setting.

Reducing cardiovascular risk in diabetes. Which factors to modify first?

Up to 80% of diabetic patients die of macrovascular complications, including CAD, stroke, and peripheral vascular disease. Because of the growing numbers of diabetic patients and the increased mortality after their first cardiovascular event, it is critical to identify and treat risk factors early and aggressively in these patients. Numerous studies in patients with type 2 diabetes have shown the benefits of aggressive treatment of blood pressure and lipids to levels that 10 years ago would have seemed abnormally low. The downward changes in "normal" limits can be frustrating to primary care physicians, but advances in treatment are redefining "normal" levels required to avoid complications in this high-risk population.

Newborn screening for homocystinuria: Irish and world experience.

Newborn screening for cystathionine beta-synthase deficiency (homocystinuria; HCU) was started in the late 1960s using a bacterial inhibition assay (BIA). At least seven countries have either national or regional screening programmes; 12 programmes are known to have discontinued. The worldwide incidence of HCU is approximately 1 in 335,000 but varies from 1:65,000 (Ireland) to 1:900,000 (Japan). Methodologies include the BIA, one-dimensional or thin-layer amino acid chromatography and, more recently, tandem mass spectrometry. The BIA diagnostic cut off concentration of blood methionine varies from 67 to 270 micromol/ (10-40 mg/l) with a median of 135 micromol/l (20 mg/l). In Ireland, 25 cases of HCU from 19 families have been identified from 1.58 million newborn infants since 1971; 21 cases were detected through the screening programme. Of the four missed cases, three were breast-fed at the time of blood collection and one was pyridoxine responsive. These findings were in broad agreement with the results from five other programmes, in which approximately one in every five cases was missed by the screening programme. Early hospital discharge, low protein intake, high blood methionine cut-off concentration and pyridoxine responsiveness were all identified as contributing to missed cases.

Clinical and biochemical studies in homocystinuria.

Homocystinuria was diagnosed in 15 (0.59%) cases on screening 2560 children for aminoacidopathies. The commonest presenting features were ectopia lentis (95%) and mental retardation (86%). Other features included, dental anomalies (40%), osteoporosis (40%), behavioral problems (33%) and arachnodactyly (13%). Diagnosis was confirmed by iodoplatinate staining of one dimensional paper chromatography of urine. All the 15 cases of homocystinuria were first treated with high dose oral pyridoxine. Only one case responded to pyridoxine therapy. All the other patients were started on a low methionine, High cysteine diet with folate supplementation. Only one patient showed a complete response to dietary therapy. Nonavailability and high cost of the commercially available methionine-free, cysteine-supplemented diet and late diagnosis were responsible for the poor response in the majority of our patients.

Nutritional therapy for pregnant women with a metabolic disorder.

Nutritional therapy is essential for a normal reproductive outcome in phenylketonuric women. In homocystinuria, fetal outcome is good in women whose disorder is responsive to vitamin B6 therapy and is poor in women whose disorder is unresponsive to therapy. Pregnancy in galactosemia is rare because of the almost universal ovarian dysfunction present in female patients with this disorder. Transplantation of the fertilized ovum is a promising possibility for these women. In women with MSUD, there has been only one case of pregnancy reported to date.

Screening of congenital hypothyroidism, phenylketonuria, galactosemia, homocystinuria, and maple syrup urine disease in moderate to severe mentally retarded Chinese children.

Five-hundred and fifty one mentally retarded children from seven institutes in Northern Taiwan were screened by dried blood spot for the detection of treatable congenital metabolic diseases, including congenital hypothyroidism, phenylketonuria, homocystinuria, maple syrup urine disease and galactosemia. We found 2 children (0.36%) with congenital hypothyroidism, 1 case (0.18%) of classical phenylketonuria and two cases (0.36%) of trisomy 21 associated with autoimmune thyroiditis. The results of our investigation suggest that congenital hypothyroidism and phenylketonuria can be the factors causing mental retardation among children in Taiwan and mass neonatal screening of these treatable inborn metabolic diseases is strongly indicated for efficiently circumventing mental retardation in our community.

Health economic analysis of the Swedish neonatal metabolic screening programme. A method of optimizing routines.

A benefit-cost analysis was carried out to optimize the routines for neonatal metabolic screening. The basis of the study was provided by results of the Swedish neonatal screening programme from 1965 to 1979. During this period over one million infants were screened by the Guthrie test for phenylketonuria and galactosaemia, and for limited periods also for tyrosinaemia, homocystinuria and histidinaemia. The benefit-cost ratio was calculated for combinations of different screening tests, recall routines, and varying degrees of coverage. The largest benefit-cost ratio was obtained with combined screening for phenylketonuria and galactosaemia, using a borderline blood phenylalanine level of 0.50 mmol/L in the Guthrie test for phenylketonuria. However, the inaccuracy of this test necessitated the use of a lower blood phenylalanine level of 0.25 mmol/L and the acceptance of a lower benefit-cost ratio. An increase in the present 98% coverage of newborns by the screening programme was found to be an effective means of improving the benefit-cost ratio in the present programme.