The Human Tissue Act: implications for clinical biochemistry.

The Human Tissue Act sets out a new legal framework for the use and retention of tissues from living persons as well as the removal, retention and use of tissue and organs from the deceased. The Human Tissue Authority (HTA) has been established as the regulatory body in relation to the Act and also to give advice and guidance initially through the publication of Codes of Practice. The HTA will also license activities within the remit of the Act and will inspect to ensure compliance with the Act and licence conditions. The Act establishes consent as the essential requirement for the lawful retention and use of tissues and organs; failure to comply could result in penalties that include a custodial sentence. In addition to solid organs and tissues, the Act applies to the use of other specimens including body fluids if they contain cells. Tissue can be stored and used without consent for a number of purposes on the basis that these are integral to the general provision of clinical and diagnostic services. These include clinical audit, education or training relating to human health, performance assessment and quality assurance. In the case of research, the Act allows tissue to be used without consent, provided that the tissue is anonymized so that the researcher cannot identify from whom the material came. Linking with medical records is allowed, provided patient-identifying information is not obtained. There is a requirement to respect the wishes of any patient who specifies that they do not wish diagnostic or therapeutic samples to be kept or used for additional purposes.

Predictors of lipoprotein(a) levels in a European and South Asian population in the Newcastle Heart Project.

BACKGROUND: Understanding of the higher susceptibility of South Asians to coronary heart disease is limited. One explanation is the combination of high prevalence of insulin resistance with higher lipoprotein(a) levels. MATERIALS AND METHODS: Lipoprotein(a) levels and genotypes in three South Asian groups aged 25-74 years (Indian, Pakistani, Bangladeshi) were compared with a European population in a cross-sectional study. Biochemical measurements included lipids, apolipoprotein A1 and B, glucose, insulin and fibrinogen. Insulin sensitivity was calculated using the homoeostasis model assessment method (HOMA). RESULTS: There was no significant difference in lipoprotein(a) levels between South Asian and European men. South Asian women combined had higher lipoprotein(a) levels than European women, a difference probably resulting from higher lipoprotein(a) levels in Pakistani women compared with Indian and Bangladeshi women. Fasting insulin and HOMA were negatively associated with Lp(a) in South Asians though the associations were statistically significant only in men. There were only modest associations between most cardiovascular risk factors and Lp(a). Twenty-seven apolipoprotein(a) size alleles were detected in the three South Asian groups ranging from 16 to 43 kringle-IV repeats. The apolipoprotein(a) size polymorphism explained 23% of the variability in lipoprotein(a) levels in South Asians. CONCLUSIONS: There were few nongenetic predictors of lipoprotein(a) levels in South Asians and Europeans. The lack of difference in Lp(a) between the South Asian and European men and the fact that differences between the women seemed to be confined to the Pakistani group offer little support to the hypothesis that higher Lp(a) levels contribute to the increased risk of heart disease in South Asians. Our findings do not support the hypothesis that susceptibility to heart disease in South Asians results from a combination of high insulin resistance and high Lp(a) levels.

Standardised monitoring of patients on long-term medication in primary care.

A three-year pilot study was initiated in collaboration with three general practices to develop a standardised monitoting (SM) system to ensure that chronically sick patients on long-term medication, such as thyroxine, diuretics angiotensin convening enzyme (ACE) inhibitors statins and antirheumatics were reliabily monitored following fixed protocols. A high standard of care was achieved, which included identifying and following up patients with borderline or unacceptable results and persistent non-attendees. In addition, the scheme guaranteed that a current set of results was provided in time for clinical review.

Lipoprotein composition and serum apolipoproteins in normoglycaemic first-degree relatives of non-insulin dependent diabetic patients.

Cardiovascular disease is the leading cause of death in non-insulin dependent diabetes mellitus and first degree relatives of such patients are at increased risk of developing diabetes and cardiovascular disease. The aim of the present study was to determine whether lipid abnormalities occur in normoglycaemic relatives of non-insulin dependent diabetic patients. Cholesterol, triglycerides, apolipoprotein A-I and apolipoprotein B concentrations were measured in serum; the lipoprotein fractions very low density, intermediate density, low density and high density lipoprotein were prepared by sequential flotation ultracentrifugation and their composition investigated. The groups were matched for age, sex and blood glucose concentrations although the relatives (n = 126) were more insulin resistant as determined using the homeostasis model assessment method [1.9 (0.8-9.0) vs 1.6 (0.4-4.9) mmol/mU per l (mean [95% confidence intervals]); p < 0.001] and had greater body mass indices [26.6 (4.1) vs 24.8 (3.9) (mean [S.D.]); p = 0.001] than control subjects (n = 126). Relatives had higher serum apolipoprotein B concentrations than control subjects [0.9 (0.3) vs 0.8 (0.3) g/l, p = 0.02) and lower serum apolipoprotein A-I concentrations (1.4 (0.3) vs 1.5 (0.3), p = 0.02). In multivariate linear regression analysis of all subjects log insulin resistance (p = 0.0001), age (p = 0.002) and waist:hip ratio (p = 0.01) were independent predicators of apolipoprotein B concentrations while waist:hip ratio (p < 0.001) and smoking status (p = 0.002) were independent predictors of apolipoprotein A-I concentrations. Lipoprotein composition (measured in a subgroup of 76 control subjects and 88 relatives), serum cholesterol and serum triglyceride concentrations did not differ between the groups. We conclude that atherogenic apolipoprotein abnormalities occur in normoglycaemic relatives of non-insulin dependent diabetic patients.

The effect of growth hormone replacement therapy for up to 12 months on lipoprotein composition and lipoprotein(a) in growth hormone-deficient adults.

The effect of growth hormone replacement therapy in near physiological doses on lipoprotein composition and serum lipoprotein(a) concentrations was investigated in growth hormone-deficient subjects. A randomised, double-blind, placebo-controlled trial of recombinant growth hormone was undertaken for 6 months followed by an open extension for a further 6 months (0.125 IU/kg per week for the first 4 weeks of each 6 month period and thereafter 0.25 IU/kg per week). A total of 18 patients with isolated growth hormone deficiency or hypopituitarism were studied. Lipid concentrations were estimated in lipoprotein fractions and protein concentrations were measured in low density lipoprotein (LDL). Glucose and glycated haemoglobin in blood and insulin, cholesterol, triglyceride, apolipoproteins A-I and B and lipoprotein(a) concentrations were measured in serum. In the placebo-controlled phase fasting blood glucose concentrations increased with growth hormone treatment from 5.0 +/- 0.2 to 5.8 +/- 0.2 mmol/l (P = 0.02) (mean +/- S.E.M.), although no significant changes were seen in lipids or lipoproteins. In the group receiving active treatment total serum cholesterol decreased from 6.0 +/- 0.4 to 5.2 +/- 0.3 mmol/l (P = 0.002) after 6 months, due to reduced LDL cholesterol concentrations. Low density lipoprotein protein concentrations fell (0.8 +/- 0.1 versus 0.7 +/- 0.1 g/l) (P = 0.005), and LDL phospholipid levels decreased from 0.9 +/- 0.1 to 0.7 +/- 0.1 mmol/l (P = 0.007). Serum cholesterol and LDL composition reverted to pre-treatment values by 12 months. Fasting blood glucose remained above pre-treatment values (P = 0.036) and fasting insulin was significantly increased (P = 0.044). There was no effect of growth hormone therapy on serum triglyceride, apolipoprotein or lipoprotein(a) concentrations. In conclusion, growth hormone therapy with near physiological doses has no long term effects on serum lipoprotein(a) concentrations or lipoprotein composition.

7-ketocholesterol, a specific indicator of lipoprotein oxidation, and malondialdehyde in non-insulin dependent diabetes and peripheral vascular disease.

Increased free radical-mediated lipoprotein oxidation may contribute to the increased prevalence of atherosclerosis in non-insulin dependent diabetes. We have determined levels of malondialdehyde (MDA) and 7-ketocholesterol, a specific indicator of free radical-mediated oxidation of lipoprotein cholesterol, in serum in very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein (LDL) and high density lipoprotein fractions of serum separated by sequential flotation ultracentrifugation. Four groups of male subjects were studied: normal controls, diabetic patients with no evidence of microvascular complications or macrovascular disease, diabetic and non-diabetic patients with peripheral vascular disease (PVD). MDA was increased in vascular disease patients (diabetic 4.5 (3.7-5.8), non-diabetic 4.4 (3.2-5.7) mumol/l, median (2.5-97.5 percentiles)) than controls (3.6 (2.9-5.0) mumol/l) (P < 0.01), but was not increased in uncomplicated diabetic patients (3.8 (3.0-4.8) mumol/l). There were no significant differences in 7-ketocholesterol concentration in LDL, but calculated total 17-ketocholesterol was lower in non-diabetic vascular patients than controls (P < 0.01). Vitamin C concentration was reduced in diabetic and non-diabetic patients with vascular disease. No significant difference in concentration of vitamin E or A was found. In six normal subjects the concentration of MDA was low in lipoproteins separated by ultracentrifugation but high in the residue following lipoprotein fractionation (70-80% total serum MDA). In conclusion, the concentration of MDA by the thiobarbituric acid assay in untreated serum may not reflect free radical damage to lipoproteins. There was no evidence of increased lipoprotein oxidation using 7-ketocholesterol in NIDDM or PVD.

Effect of storage at -70 degrees C on lipid, lipoprotein and apolipoprotein concentrations.

We have investigated the effects on lipid, apolipoprotein and lipoprotein measurements of storing unfractionated serum from normolipidaemic and hyperlipidaemic subjects at -70 degrees C for 10 days, 3 months and 6 months. Total serum concentrations of lipids and apolipoproteins were stable except for triglyceride concentrations. These increased on storage although the change was < 2.0% after 6 months. Storage of serum before sequential flotation ultracentrifugation resulted in decreased free cholesterol and phospholipid concentrations in very low density lipoproteins. In low density lipoproteins, free cholesterol concentrations increased and protein concentrations decreased on storage, while esterified cholesterol and phospholipid concentrations fell after 10 days but did not differ from baseline concentrations after storage for 6 months. Within high density lipoproteins, there were decreases in triglyceride and protein concentrations. Although storage of serum at -70 degrees C for up to 6 months did not result in extensive changes in most lipoprotein fractions, separation of lipoprotein fractions from serum should, ideally, be performed as soon as possible after collection.

Serum concentrations of vitamins A and E in impaired glucose tolerance.

Serum concentrations of vitamins A and E were measured in 32 subjects with impaired glucose tolerance (IGT) and 148 subjects with normal glucose tolerance using reversed-phase high-performance liquid chromatography. Fasting glucose, insulin and lipid concentrations were also measured. Serum vitamin A concentrations were higher in subjects with IGT 2.5 (1.1-3.4) vs. 2.1 (1.4-3.2) mumol/l [median (2.5-97.5 percentiles)] (P = 0.002), the difference remaining significant after adjustment for triglycerides (P = 0.028). There was a univariate association between vitamin A levels and insulin resistance (r = 0.164; P = 0.02) and in multivariate logistic regression analysis the relative risk of subjects with high vitamin A concentrations having IGT was 3.8 (P = 0.002). There were no differences in serum vitamin E concentrations between the groups. These data suggest that higher vitamin A concentrations found in non-insulin-dependent diabetes pre-date the onset of diabetes. Further studies are required to confirm this finding and to investigate the possibility of a role for vitamin A in the aetiology of diabetes and IGT.

Self-monitoring of triglycerides by type 2 diabetic patients: variability in fasting and postprandial levels.

Triglycerides are an important risk factor for coronary heart disease in Type 2 (non-insulin-dependent) diabetes mellitus. Although Type 2 diabetic patients have an exaggerated postprandial triglyceride response to a fat meal test, little is known about the variability of triglyceride concentrations in day-to-day life. We have studied the variability in triglyceride concentrations in 24 Type 2 diabetic patients over 6 months by having them record fasting and postprandial triglyceride concentrations at home using a Reflotron dry chemistry analyser. All patients were able to use the analyser effectively, with a correlation of 0.97 between patients' monthly Reflotron readings and those recorded by the laboratory. Over 1600 measurements were performed. The results demonstrate a large variation in both fasting (median 1.95 mmol l-1, range 0.8-6.7 mmol l-1) and postprandial triglyceride concentrations (median 2.68 mmol l-1, range 0.8-6.7 mmol l-1). This variation was accounted for by both a large intra- and inter-individual variation. Although there was a strong correlation overall between fasting and postprandial triglyceride concentrations r = 0.925 (p < 0.001), this did not apply on an individual basis. In conclusion, the large variability in triglyceride concentrations should be considered before introducing pharmacological therapy for hypertriglyceridaemia in Type 2 diabetes mellitus.

Lipoprotein (a) concentrations and apolipoprotein (a) phenotypes in normoglycaemic relatives of type 2 diabetic patients.

Serum lipoprotein (a) concentrations (Lp(a)) are largely under genetic control, and are strong predictors of coronary heart disease. It has been hypothesised that Lp(a) may contribute to the increased risk of coronary heart disease in familial Type 2 diabetes mellitus. We therefore examined the Lp(a) concentrations and the apolipoprotein (a) (apo(a)) phenotypes in 126 normoglycaemic first degree relatives from families with two or more living Type 2 diabetic patients. These were compared with 147 sex matched normoglycaemic control subjects with no family history of diabetes. Lp(a) concentrations were measured using an enzyme-linked immunosorbent assay (ELISA), and apo(a) isoforms were determined and classified according to the relative mobility of apo(a) on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), relative to that of apolipoprotein B-100. There were no significant differences in Lp(a) concentrations between the relatives (R) and controls (C): 11.2 (R) vs. 11.1 (C) mg/dl (median). The distribution of apo(a) phenotypes was not significantly different between groups 0.65 (R) vs. 0.67 (C). These results show that first degree relatives at risk of developing Type 2 diabetes do not have abnormal Lp(a) concentrations or apo(a) phenotypes.

Measurement of sugar probes in serum: an alternative to urine measurement in intestinal permeability testing.

The percentage dose of lactulose and mannitol excreted in urine after oral ingestion is used as a noninvasive method of assessing small intestinal permeability. The collection of incomplete or inaccurately timed urine samples can lead to errors in estimation of sugar probe molecules. We describe an HPLC method for the simultaneous determination of lactulose and mannitol in serum after oral ingestion of test sugars. We applied the test to healthy volunteers and to subjects undergoing jejunal biopsy for suspected gluten-sensitive enteropathy. The ratio of concentrations of lactulose and mannitol in serum discriminated well between subjects with a normal biopsy and those with villous atrophy, discrimination being best at 90 min postdose. The results agree well with lactulose:mannitol ratios determined in urine (r= 0.88), and the two methods can be used interchangeably. The determination of mannitol and lactulose in serum provides an acceptable alternative to urine collection and may be particularly useful in young children. It also reduces the time spent on the investigation from 5 h to 90 min.

Assessing the impact of blood sample type on the estimated prevalence of impaired glucose tolerance and diabetes mellitus in epidemiological surveys.

In a prospective study of 353 patients who had undergone coronary artery bypass graft surgery a 75 g oral glucose tolerance test was performed at 3 months and 12 months after surgery. Venous whole blood glucose and venous plasma glucose samples were assayed on a bench-top analyser. The World Health Organization diagnostic criteria for impaired glucose tolerance and diabetes mellitus were applied to venous whole blood glucose and venous plasma glucose measurements. The difference between the plasma and whole blood concentration of glucose was 0.79-0.86 mmol l-1 at a plasma glucose of 7.8 mmol l-1 and 1.22-1.24 mmol l-1 at a plasma glucose of 11.1 mmol l-1 (compared to 1.1 mmol l-1 by World Health Organization criteria at both cut-points). Despite this, in our subject population with a high prevalence of impaired glucose tolerance (20.3% at 3 months, 15.3% at 12 months) and diabetes mellitus (10.1% at 3 months and 12 months), there was no significant difference in the proportion classified impaired glucose tolerance or having diabetes mellitus using venous whole blood glucose compared to venous plasma glucose. We conclude that despite the minor anomaly between WHO diagnostic criteria based on venous whole blood glucose and venous plasma glucose measurements, these criteria are robust and give broadly comparable population prevalence of diabetes mellitus and impaired glucose tolerance irrespective of blood sample choice.