The association of birthweight and contemporary size with insulin resistance among children from Estonia and Denmark: findings from the European Youth Heart Study.

OBJECTIVES: To assess the associations of birthweight, contemporary body mass index and height with insulin resistance in children. DESIGN: Cross-sectional study. PARTICIPANTS: From Estonia (n = 1174) and Denmark (n = 1018), 2192 school children aged 9 and 15 years were randomly selected. MAIN OUTCOMES: Insulin resistance (homeostasis model assessment), triglyceride levels, high-density lipoprotein cholesterol and systolic blood pressure. RESULTS: There was an inverse association between birthweight and insulin resistance and a positive association between contemporary body mass index and insulin resistance. With adjustment for maternal and paternal educational level, income, smoking and body mass index, an increase of one unit of sex, age and country standardized body mass index z-score was associated with a 5% (95% CI: 2, 7%) increase in homeostasis model assessment (HOMA) score and a one-unit z-score increase in birthweight with a 2% (95% CI: 0, 5%) decrease in HOMA score. In the 9-year-old age group, height was positively associated with insulin resistance [for a one-unit increase in height z-score HOMA score increased by 30% (95% CI: 14, 50%)], but in the 15-year-old age group there was no association between height and insulin resistance (4% (95% CI: -5, 14%), P for interaction with age group = 0.001). For both ages, those in the lowest third of the birthweight distribution and highest third of the body mass index distribution were most insulin resistant and, among 9-year olds, those in the lowest third of the birthweight distribution and highest third of the height distribution were most insulin resistant. Birthweight was only inversely associated with systolic blood pressure when adjustment was made for either contemporary body mass index or height and there was no association between birthweight and high-density lipoprotein or triglyceride concentrations. CONCLUSIONS: Taken together, these results suggest that a slow intrauterine growth trajectory and/or a fast post-natal growth trajectory is associated with greater insulin resistance in childhood.

Infant feeding and components of the metabolic syndrome: findings from the European Youth Heart Study.

AIMS: To assess the associations of type and duration of infant feeding with components of the metabolic syndrome in children aged 9 and 15. METHODS: A total of 2192 randomly selected schoolchildren aged 9 and 15 years from Estonia (n = 1174) and Denmark (n = 1018) were studied. Insulin resistance (homoeostasis model assessment), triglyceride levels, high density lipoprotein cholesterol, and systolic blood pressure were measured. RESULTS: Children who had ever been exclusively breast fed had lower systolic blood pressures than those who were not. With full adjustment for age, sex, country, birth weight, pubertal stage, body mass index, height, maternal and paternal education, income, smoking, and body mass index the mean systolic blood pressure of children who had ever been breast fed was 1.7 mm Hg (95% CI -3.0 to -0.5) lower than those who had never been exclusively breast fed. There was a dose-response in this association with decreasing mean systolic blood pressure across categories from never exclusively breast fed to breast fed for more than six months. Exclusive breast feeding was not associated with other components of the metabolic syndrome. Results were similar when examined separately in each country. CONCLUSIONS: The magnitude of the association, its independence of important confounding factors, and the dose-response suggest that exclusive breast feeding is causally associated with reduced systolic blood pressure. The magnitude of the effect we found with blood pressure is comparable to the published effects of salt restriction and physical activity on blood pressure in adult populations, suggesting that it is of public health importance.

Assessment of homocysteine as a cardiovascular risk factor in clinical practice.

Elevated plasma total homocysteine concentrations are a marker of vitamin deficiency and a risk factor for cardiovascular disease. It is possible that vitamin supplementation with folic acid and other B vitamins, which lower plasma homocysteine concentrations, may reduce the risk of cardiovascular disease. Large-scale clinical trials are currently underway to assess the homocysteine hypothesis of cardiovascular disease. Pending the outcome of such trials, measurement of plasma homocysteine concentrations in people at high risk of cardiovascular disease may help to identify patients who could benefit from more intensive treatment of classical cardiovascular risk factors. The introduction of immunoassays for homocysteine determination has made assessment of homocysteine status accessible to most routine hospital laboratories, and this review summarizes the evidence on why and how to assess homocysteine as a risk factor for cardiovascular disease in clinical practice.

Gene-environment and gene-gene interaction in the determination of plasma homocysteine levels in healthy middle-aged men.

Healthy middle-aged men (n = 1,470) from eight general practices across Britain were examined for plasma total homocysteine levels and genotyped for the A222V polymorphism in the methylene-tetrahydrofolate (MTHFR) gene, the 68 bp insertion polymorphism in exon 8 of the cystathionine b synthase (CBS) gene and the D919G polymorphism in the methionine synthase (MS) gene. The median value for plasma homocysteine was 11.90 micromol/l (25-75% Interquartile range 10.10-14.20) for the whole sample. Smokers had significantly higher homocysteine levels than non-smokers (12.90 vs 11.70 micromol/l and p < 0.00005) and levels significantly differed according to folate (p-value < 0.00005), with men in the lowest quartile of folate having the highest median homocysteine levels. Genotype at all three loci was associated with differences in plasma homocysteine level. Individuals homozygous for the MTHFR V222 allele had 1.6 micromol/l higher median homocysteine levels when compared to the other two genotypes (p < 0.00005), while for the CBS and MS genes, individuals carrying one or more of the rare alleles had lower median homocysteine than individuals homozygous for the common allele (0.80 micromol/l, p < 0.03, and 0.70 micromol/l, p < 0.04 respectively). The raising effect associated with homozygosity for the V222 allele was greater in men in the lowest quartile of folate (interaction between folate and genotype p = 0.02), but none of the genotype effects was significantly modulated by B12 levels. While the raising effects of V222 and MS D919 homozygosity on homocysteine level were essentially additive, the homocysteine lowering effect associated with the CBS 68bp allele was seen most strongly in men homozygous for the V222 allele (MTHFR-CBS genotype interaction p = 0.03) and the D919 allele (MS-CBS interaction p = 0.09). Age, folate, B12 and smoking explained 13.48% of the variance while the three genotypes combined and with interaction terms explained only an additional 2.63%. This interaction between CBS genotype and MTHFR and MS genotype points to a key role of the CBS transulphuration pathway in the metabolism of homocysteine that may be particularly important as a compensatory mechanism in subjects with low dietary folate.

Cost-effective strategy for the serological investigation of coeliac disease.

Increased numbers of requests for serological investigation of coeliac disease, and a local trend to request both anti-gliadin antibodies (AGA) and anti-endomysium antibodies (AEA) simultaneously, resulted in cost pressures that prompted a review of our practice. Serology results from all patients (771 children, 511 adults) investigated for coeliac disease over a 3-year period were compared with small intestine histology where available. IgG AGA and IgA AGA were measured by enzyme-linked immunosorbent assay (in-house), IgA AEA by immunofluorescence (send-away contract). Overall diagnostic performance was as follows: AGA sensitivity 84%, specificity 88%, positive predictive value (PPV) 24%, negative predictive value (NPV) 99%; AEA sensitivity 88%, specificity 97%, PPV 65%, NPV 99%. Results showed AGA, with its high NPV, to be a suitable first-line test to exclude coeliac disease. The high specificity of AEA makes it a suitable confirmatory test when AGA is positive. Introduction of this step-wise approach to coeliac disease investigation resulted in cost savings of at least Pound Sterling 5000 per year without detriment to the clinical service.

C677T (thermolabile alanine/valine) polymorphism in methylenetetrahydrofolate reductase (MTHFR): its frequency and impact on plasma homocysteine concentration in different European populations. EARS group.

A common polymorphism has been described in the methylenetetrahydrofolate reductase (MTHFR) gene, substituting an alanine (A) for a valine (V), where the V allele results in a thermolabile enzyme with reduced activity. This polymorphism is easily detectable by PCR amplification and digestion with HinfI restriction enzyme. We describe the use of the MADGE high throughput genotyping system for rapid typing of this polymorphism. Seven hundred and eighty five individuals participating in the European Atherosclerosis Research Study II (EARS II), aged 22-25 from 14 universities in 12 countries across Europe were genotyped for this polymorphism. The frequency of the V allele was 0.32 overall (95% CI; 0.30-0.35), but was significantly lower in the Baltic countries (0.23; 95% CI; 0.19-0.28) compared with the other regions of Europe (0.37; 95% CI; 0.32-0.38) (P < 0.001). Individuals homozygous for the V allele had statistically significant (P < 0.001) higher plasma homocysteine (16.5 micromol/l) compared with those heterozygous for an A allele (10.4 micromol/l) or homozygous for an A allele (10.0 micromol/l). This effect was seen in all countries and regions of Europe. Mean plasma homocysteine levels were significantly higher in the South compared to the Baltic, UK and Middle regions (P = 0.001), but this difference was not explained by the difference in the frequency of the V allele in the samples. This polymorphism explained 12.3% of the total sample variance in plasma homocysteine, other measured factors (smoking, alcohol consumption, systolic blood pressure, physical activity) explained 0.7%. This study demonstrates the large and consistent impact of the thermolabile MTHFR variant on plasma homocysteine levels in different European populations, and shows a regional difference in the levels of homocysteine that must be explained by other genetic or environmental factors.

Linkage disequilibrium at the cystathionine beta synthase (CBS) locus and the association between genetic variation at the CBS locus and plasma levels of homocysteine. The Ears II Group. European Atherosclerosis Research Study.

Cystathionine beta synthase (CBS) is a key enzyme in homocysteine metabolism. We have examined four apparently non-functional polymorphisms in the CBS gene and have determined their frequency, degree of linkage disequilibrium and association with plasma homocysteine levels. The polymorphisms are a 68 bp insertion in exon 8, C699T in exon 8, C1080T in exon 11 and C1985T in the 3' untranslated region. 785 individuals participating in the European Atherosclerosis Research Study II (EARSII), from 11 countries across Europe were genotyped for these polymorphisms. The 68bp insertion had the highest frequency in the UK and in the Middle region, with a lower frequency in the Baltic and the South (p = 0.01), and the exon 11 polymorphism had the highest frequencies of the rare allele in the Baltic (p < 0.05). There was a high degree of linkage disequilibrium between the polymorphisms (p < 0.001 overall), except between C699T and the C1985T, with three common haplotypes accounting for nearly 80% of chromosomes. Examination of the association between these polymorphisms and plasma homocysteine levels revealed that the carriers of the rare alleles of the C699T, C1080T and C1985T polymorphisms had lower plasma homocysteine concentrations than those homozygous for the common alleles, although these differences were not statistically significant. The thermolabile valine variant caused by a substitution of a C for a T at nucleotide 677 in the methylenetetrahydrofolate reductase (MTHFR) has previously been shown to have profound effects on plasma levels of homocysteine in this sample, but the homocysteine-raising effect associated with this thermolabile variant was not seen in carriers of the 68 bp insertion, with this interaction being statistically significant (p < 0.001). These data demonstrate that variation in the CBS gene as detected with these four polymorphisms, had no statistically significant effect on plasma homocysteine levels in these healthy young men. However, the presence of the 68 bp insertion, which is found in approximately 7.5% of individuals in the populations of Europe sampled, abolishes the raising effect of thermolabile MTHFR Val/Val genotype, and may be of importance in the situation of high homocysteine.

Effect of simvastatin therapy on cell membrane cholesterol content and membrane function as assessed by polymorphonuclear cell NADPH oxidase activity.

Cell membrane cholesterol is an important determinant of membrane fluidity. Changes in fluidity have important consequences for membrane function. Treatment of hypercholesterolaemia could therefore affect membrane function by reducing cell membrane cholesterol levels. The aim of this study was to determine whether treatment with simvastatin affects membrane cholesterol and the activity of the polymorphonuclear cell membrane enzyme NADPH oxidase. Blood was obtained from 12 hypercholesterolaemic patients before, and 6 weeks after, treatment with simvastatin, and from 20 normolipidaemic subjects. Cell cholesterol was in the unesterified from indicating that it was membrane-associated. Pre-treatment mean cell cholesterol concentration in the hyperlipidaemics was higher (P < 0.05) than in the normolipidaemics [4.19 fmol/cell, 95% confidence interval (CI) 3.38-5.05 versus 3.10 fmol/cell, 95% CI 2.58-3.61]. There was a strong correlation between cell cholesterol content and NADPH oxidase lag phase (R(s) = 0.76, P < 0.01). Cell cholesterol fell to 3.52 fmol/cell (95% CI 2.77-4.28, P < 0.05) following treatment and there was a correlation (R(s) = 0.61, P < 0.05) between the reductions in cell cholesterol and lag phase.

Effect of concentrated red grape juice consumption on serum antioxidant capacity and low-density lipoprotein oxidation.

This study examines whether the beneficial antioxidant effects of red wine can be reproduced by nonalcoholic red grape juice concentrate. Seven subjects consumed 125 ml concentrate daily for 7 days. Following first ingestion there was a rise in serum total antioxidant capacity (TAC) from 441 to 478 mumol/l at 60 min (p < 0.005). On day 8, TAC was 50 mumol/l higher than at baseline (p < 0.05). There was reduced susceptibility of low-density lipoprotein (LDL) to oxidation. Red grape juice concentrate ingestion results in increased serum antioxidant capacity and protection of LDL from oxidation and thus nonalcoholic red grape extract may have similar beneficial effects to red wine.

Hyperhomocysteinaemia is a risk factor for vein graft stenosis.

OBJECTIVES: Many infrainguinal vein graft failures are due to progressive vein graft stenosis (VGS) from intimal hyperplasia. Systemic factors have been implicated in the aetiology of intimal hyperplasia. Hyperhomocysteinaemia (HHCA) is established as an independent risk factor for coronary and peripheral arterial disease. The objective of this study was to examine the influence of HHCA and other serological factors upon the development of VGS. STUDY DESIGN: Thirty-eight patients who had undergone infrainguinal vein bypass were recruited to a case/control study from a graft surveillance program. Nineteen patients with documented VGS were matched against controls without stenosis for age, sex, length of time from surgery, diabetes, smoking history and preoperative symptom score. All patients were recalled for Duplex ultrasound scans, venesection and carbon monoxide estimation which were performed in a blinded fashion. RESULTS: Statistical analysis of all parameters revealed that plasma homocysteine was significantly elevated in patients with VGS (p < 0.3, Wilcoxon rank sum). CONCLUSIONS: These results suggest that HHCA is a previously unidentified risk factor for VGS. Patients with HHCA are susceptible to VGS and preoperative investigation would allow identification of patients at risk.

Homocysteine: an independent risk factor for the failure of vascular intervention.

Hyperhomocysteinaemia is an independent risk factor for the early development of arterial disease. Homocysteine and cardiovascular risk factors were assessed in 41 young and 25 older patients with vascular disease. As homocysteine may act by the generation of free radicals, total antioxidant capacity was measured. Hyperhomocysteinaemia was found in 29 per cent of patients but there was no difference between young and older patients. Homocysteine level was unrelated to other cardiovascular risk factors. Young age, diabetes and hyperhomocysteinaemia were independent risk factors for the failure of vascular procedures (P = 0.006). Patients with hyperhomocysteinaemia had raised total antioxidant capacity. The potential of identifying and treating a subgroup of patients with a poor prognosis deserves further study.

Defect in the lipoyl-bearing protein X subunit of the pyruvate dehydrogenase complex in two patients with encephalomyelopathy.

Among the many metabolic encephalomyelopathies caused by deficiencies in the pyruvate dehydrogenase complex (PDHC), nearly all involve its E1 subunit. We describe two new familial cases of PDHC deficiency with encephalomyelopathy, chronic lactic acidemia, and a normal E1 subunit of PDHC but deficiency in another component. Activity of PDHC was measured in cultured skin fibroblasts and skeletal muscle, and immunoblot studies were performed on mitochondrial extracts from skin fibroblasts. Spectra of muscle tissue, obtained in vivo with phosphorus 31 nuclear magnetic resonance, were recorded both at rest and with exercise. The PDHC activity was markedly reduced to 10% to 20% of normal values in both cultured skin fibroblasts and skeletal muscle. Immunoblotting of skin fibroblast mitochondrial extracts showed a specific deficiency in the protein X component of PDHC but normal E1, E2, and E3 components. Spectra obtained with 31P nuclear magnetic resonance showed alterations compatible with those found in mitochondrial myopathies. This is the second description of an encephalomyelopathy associated with a specific absence of the lipoyl-containing protein X component, which has a structural role in the formation of a functional PDHC.

Suppression of lipolysis in normal man does not inhibit recovery from insulin-induced hypoglycaemia.

Pharmacological suppression of lipolysis is being increasingly used in the treatment of diabetic hyperlipidaemia. Although theoretical hazard of such treatment is that recovery from hypoglycaemia might be impaired. Seven normal subjects were therefore studied on two occasions, following treatment with a single dose of either acipimox 250 mg or placebo. Hypoglycaemic recovery was unaffected, despite effective suppression of plasma non-esterified fatty acid levels with acipimox. The results suggest that under these conditions activation of lipolysis may not be essential to recovery from hypoglycaemia.

Do in-vivo suppression and stimulation of lipolysis influence thyroid hormone levels in man?

To determine whether increases in non-esterified fatty acids alter free thyroid hormone and TSH levels, the effect of endogenous activation of lipolysis by insulin-induced hypoglycaemia was examined in seven healthy volunteers pretreated with placebo or acipimox. Whilst levels of non-esterified fatty acid were very different in the two groups, levels of free thyroxine, tri-iodothyronine and TSH were unchanged. Thus, within the range of non-esterified fatty acid levels likely to be seen in clinical practice, any effect on thyroid hormone measurements can be safely ignored.

Neonatal parenteral nutrition with a fat emulsion containing medium chain triglycerides.

Fifty-one newborn infants requiring parenteral nutrition were randomly assigned to receive a 50% medium chain triglyceride/50% long chain triglyceride lipid emulsion or the conventional 100% long chain triglyceride emulsion. Fat was administered daily for 20 hours, to a maximum of 3 g/kg/day. Plasma triglycerides, cholesterol, free fatty acids, ketones, glucose and capillary blood gases were monitored daily up to the sixth day of fat infusion. There were no significant differences in mean plasma triglycerides and free fatty acids between the two groups. No cases of hyperketonaemia were detected in the infants studied. Hyperglycaemic episodes were detected with similar frequency in both groups. The group who received the emulsion containing medium chain triglycerides had significantly lower mean plasma cholesterol values during the study. After 6 days of intravenous fat administration mean plasma cholesterol was more than 100% higher in the group which received the conventional emulsion. Differences in cholesterol content between the emulsions and a cholesterol lowering effect of medium chain triglycerides are possible explanations for these findings.

Incidence of familial hypercholesterolaemia in premature deaths due to coronary heart disease.

Cholesterol estimations were performed on blood collected postmortem from a group of subjects coming to autopsy and showing that they had had a high risk for hypercholesterolaemia, and from an unselected group of subjects dying in hospital. Subjects from the first group had apparently been healthy, aged less than 45 years, had had no known risk factors for hyperlipidaemia, and showed extensive coronary artery atheroma (stenosis greater than 50% by diameter). Eleven cases from 485 consecutive autopsies fulfilled these criteria; three showed considerable hypercholesterolaemia (11.2, 11.8, and 21.6 mmol/L). Family studies confirmed the diagnosis of familial hypercholesterolaemia in one case; the other two remain unproven. Cholesterol measurement by cholesterol oxidase and quinoneimine dye production is subject to interference by haemolysis; provided that serum haemoglobin is less than 200 mg/dl, the cholesterol underestimate is less than 5%. The decline in serum cholesterol in the group of unselected subjects was 1.7 (0.3-4.9) mmol/L, 50.4 (28-84) h postmortem. Results are means and ranges for seven subjects. Measurement of cholesterol in serum obtained postmortem (provided that the sample is not grossly haemolysed) is a valid approximation of antemortem levels: this measurement should be made when autopsy reveals evidence of premature coronary heart disease. If hypercholesterolaemia is discovered, the diagnosis of familial hypercholesterolaemia, a common genetic disorder inherited in an autosomal dominant fashion, should be considered and appropriate family studies instituted.