What is the best predictor of the atherogenic LDL subclass phenotype 'pattern B' in patients with type 2 diabetes mellitus?

BACKGROUND: The atherogenic lipoprotein phenotype 'pattern B' comprises a predominance of small-dense low-density lipoprotein (sdLDL). Gradient gel electrophoresis (GGE) is considered a 'gold standard' method for identifying this phenotype, but is impractical for routine laboratory use. The low-density lipoprotein cholesterol:apolipoprotein-B (LDL-C:Apo-B) ratio has been advocated as a surrogate marker for sdLDL and a direct assay for sdLDL has recently become available. We compared the sdLDL assay and LDL-C:Apo-B with more established lipid parameters to predict the presence of 'pattern B' phenotype. METHOD: Blood was collected from 97 fasted subjects on three separate occasions. Total cholesterol, triglyceride, Apo-B and sdLDL were measured; LDL- and HDL-cholesterol were determined after ultracentrifugation. The predominant LDL particle size and phenotype were assigned by GGE. RESULTS: 'Pattern B' phenotype was identified in 36% of samples. Peak particle size showed a positive correlation with HDL-cholesterol and a negative correlation with triglyceride and Apo-B. Receiver operating curve (ROC) analysis showed triglyceride:HDL-C ratio and triglyceride alone to be the best predictors of 'pattern B' phenotype, with area under the curve (AUC) being 0.87 and 0.84, respectively. AUCs for sdLDL (0.74) and LDL-C:Apo-B (0.71) were significantly lower (P < 0.05). A high sdLDL concentration had the greatest specificity (95%) and positive predictive value (74%) for 'pattern B' phenotype, but low sensitivity (43%). CONCLUSION: Direct measurement of sdLDL provided the most specific predictor of 'pattern B' phenotype, whereas triglyceride:HDL-C ratio or triglycerides alone, parameters readily available in most laboratories, were the best predictors by ROC analysis.

Pseudo-hypertriglyceridaemia: a measurement artefact due to glycerol kinase deficiency.

A man presented with elevated plasma triglycerides and was commenced on fibrate treatment. The triglycerides did not fall and compliance was questioned. The triglyceride elevation was inconsistent with the observed lack of turbidity in the plasma sample. Triglyceride elevation was not confirmed by a different analytical method and lipoprotein electrophoresis showed a normal very low density lipoprotein (VLDL) band pattern. Glycerol kinase deficiency was suspected and was supported by elevated urine glycerol, and confirmed by reduced leucocyte enzyme activity and mutational analysis of the GK gene which showed a novel three base pair deletion. Demonstration of a point mutation also excludes a contiguous gene deletion syndrome.

The effect of Helicobacter pylori infection and dietary iron deficiency on host iron homeostasis: a study in mice.

BACKGROUND: Helicobacter pylori, which requires iron to survive, may cause host iron deficiency by directly competing with the host for available iron or by impairing iron uptake as a consequence of atrophy-associated gastric hypochlorhydria. The aim of this study was to examine the effect of H. pylori infection and dietary iron deficiency on host iron homeostasis in a mouse model. MATERIALS AND METHODS: H. pylori SS1-infected and uninfected C57BL/6 mice, fed either a normal diet or an iron-deficient diet, were assessed for iron status and infection-associated gastritis over a 30-week period. RESULTS: After 10 weeks, serum ferritin values were higher in H. pylori-infected mice than in uninfected controls, irrespective of dietary iron intake (p = .04). The infection-related increase in body iron stores persisted in the iron-replete mice but diminished over time in mice with restricted dietary iron intake (p < .0001). At 30 weeks serum ferritin levels were lower in these animals (p = .063). No significant difference in bacterial numbers was detected at the 30-week time point (p > .05) and the histological changes observed were consistently associated with infection (p < .01) and not with the iron status of the mice (p = .771). CONCLUSIONS: Infection with H. pylori did not cause iron deficiency in iron-replete mice. However, diminished iron stores in mice as a result of limited dietary iron intake were further lowered by concurrent infection, thus indicating that H. pylori competes successfully with the host for available iron.

Haemoglobin Marseille-Long Island and interpretation of HbA1c: which HbA1c result is the "right answer"?

A woman was screened for diabetes using glycated haemoglobin (HbA1c). Vastly different results were obtained by high performance liquid chromatography (45%), immunoassay (2.9%), and affinity chromatography (4.2%) compared with the non-diabetic range of less than 6.4%. Mass spectral studies confirmed the presence a haemoglobin variant, haemoglobin Marseille-Long Island which had confounded interpretation by all methods.

Haemochromatosis gene mutations Cys282Tyr and His63Asp are not increased in Type 2 diabetic patients compared with the Canterbury (New Zealand) general population.

Genetic predisposition to haemochromatosis may be an important aetiological factor in some cases of Type 2 diabetes. Our aim was therefore to test the hypothesis that the haemochromatosis gene mutations Cys282Tyr and His63Asp are more prevalent in Type 2 diabetic patients compared with the Canterbury, New Zealand general population. We studied 230 consecutive patients referred to the Diabetes Services with age > or = 30 years and considered to have Type 2 diabetes. DNA was extracted from whole blood and amplified by polymerase chain reaction prior to restriction fragment length polymorphism analysis. The frequency of the mutations was compared with that observed previously in 1064 subjects from the Canterbury general population by chi2 testing. Iron was measured by a colorimetric method, transferrin by rate nephelometry and ferritin by immunoassay. There were 2/230 (0.8%) Cys282Tyr homozygous subjects in the diabetic group compared with 5/1064 (0.5%) NS in the general population. Although there was a trend to lower incidence of Cys282Tyr heterozygosity in the diabetic group, there was no significant difference for any of the six genotype frequencies between the two groups. Haemochromatosis gene mutations Cys282Tyr and His63Asp are therefore not increased in Type 2 diabetics compared with the general population. Transferrin saturation was a sensitive marker (100%) of genetic haemochromatosis, although ferritin had low specificity (77.8%). Genetic susceptibility to haemochromatosis is not an important aetiological factor for diabetes, and targeted screening of diabetic patients for haemochromatosis is not indicated.

The significance of haemochromatosis gene mutations in the general population: implications for screening.

BACKGROUND: Haemochromatosis is associated with mutations in the HFE gene but the significance of these mutations in the general population is unknown. AIMS: To determine the frequency of HFE gene mutations in the general population, their effect on serum iron indexes, and their role in screening for haemochromatosis. METHODS: Deoxyribonucleic acid (DNA) from 1064 randomly selected subjects was analysed for the C282Y and H63D mutations in the HFE gene. Serum iron, transferrin saturation, and ferritin were measured and individuals with increased iron indexes were investigated to confirm or exclude a clinical diagnosis of haemochromatosis. RESULTS: Mutations were identified in 409 individuals (38.4%) with heterozygote (carrier) frequencies of 13.2% and 24.3% for the C282Y and H63D mutations respectively. Heterozygosity for either mutation significantly increased serum iron and transferrin saturation but despite a similar trend for ferritin, this was only significant for C282Y homozygotes. Five individuals (0.47%) were homozygous for the C282Y mutation, three of whom had haemochromatosis confirmed by liver biopsy (0.28%). The other two C282Y homozygotes would not have been detected by phenotypic screening alone. CONCLUSIONS: HFE mutations are present in 38.4% of the population, affect serum iron indexes, and are important determinants of iron status. The population frequency of genetically defined haemochromatosis (C282Y homozygosity) is approximately one in 200 and is higher than the prevalence of clinically apparent haemochromatosis.

Distribution of blood lead levels in a birth cohort of New Zealanders at age 21.

Little is known about lead exposure in the general population of young adults. In this study, whole blood lead concentration (PbB) was determined in a sample of the Dunedin Multidisciplinary Health and Development Study, a well-documented birth cohort of New Zealanders aged 21 years in 1993-1994. PbB in those who consented to venipuncture at 21 years of age (n = 779; 411 males, 368 females) was compared to PbB for the same cohort at age 11 years. The PbB at age 21 ranged from 0.4 to 56 micrograms/dl with a geometric mean of 4.5 micrograms/dl (95% CI, 4.3-4.7 micrograms/dl). Only three individuals had a PbB above 30 micrograms/dl. Males had significantly higher PbB than females (geometric mean 6.0 vs. 3.2 micrograms/dl; p < 0.0001). The PbB at age 21 was 53% lower than in the same individuals at age 11 (geometric mean 4.8 vs. 10.2 micrograms/dl; p < 0.001; n = 480) and the correlation between corresponding values was weak (r = 0.19; p < 0.001). PbB at age 21 showed significant associations with high risk occupational activities, recreational exposure, domicile close to a main road, smoking, and male sex. Blood lead concentrations continue to fall in New Zealand, but occupational and recreational activities remain a significant source of lead exposure.

Comparison of histological and biochemical hepatic iron indexes in the diagnosis of genetic haemochromatosis.

AIMS: To compare a histological hepatic iron index with a biochemical hepatic iron index, derived from atomic absorption spectroscopy measurements of hepatic iron content, for the diagnosis of genetic haemochromatosis (GH). METHODS: Histological sections of liver biopsy specimens from 70 subjects, who had previously had their biochemical hepatic iron index measured, were examined. The iron stores were scored to derive a histological hepatic iron index and were also graded from 0 to 4 by a standard grading system. The case history of each patient was then reviewed to establish a definitive clinical diagnosis and patients were classified as GH, non-GH or indeterminate. RESULTS: There were 26 cases of GH, 40 cases of non-GH and four indeterminate cases in whom a definite clinical diagnosis was not established. Using a biochemical hepatic iron index cut off level of 2.0, two cases were misclassified, with one case of GH having a biochemical hepatic iron index of 1.8 and one non-GH case having a biochemical hepatic iron index of 3.1. This could not have been improved by altering the cut off level. Using the recommended cut off level of 0.15, the histological hepatic iron index was raised in all cases of GH, but was also increased in 11 of the 40 non-GH patients. The specificity of this histological index can be improved by increasing the cut off level to 0.30. A histological iron grade of > or = 3 is more specific than the histological index but has a lower sensitivity, which particularly affects the diagnosis of younger patients with GH. CONCLUSIONS: The biochemical hepatic iron index is a reliable method for establishing a diagnosis of homozygous GH. In contrast, the histological hepatic iron index as originally described is non-specific and does not reliably distinguish patients with GH from others with a raised hepatic iron index due to other causes. The specificity of this index can be improved by increasing the cut off level used, but the discrimination provided by the histological index is still inferior to that provided by the biochemical hepatic iron index.

Investigation of subjects with abnormal iron studies: role of the hepatic iron index.

AIM: Genetic haemochromatosis is a common disorder resulting in increased iron deposition in the liver and other organs but can be difficult to diagnose. The aim of this study was to assess the diagnostic value of the conventional tests for iron overload (percentage saturation of transferrin, serum ferritin and grading of iron staining on liver biopsy) and compare these with the newer quantitative biochemical measurements of liver iron. METHOD: A retrospective analysis was made of 108 consecutive patients referred for quantitative liver iron measurements. Iron studies were obtained in 66 of the 108 subjects of whom 60 had abnormal screening tests defined as percent saturation of transferrin (> 60%) and/or ferritin > 350 micrograms/L for females and > 450 micrograms/L for males. Based on clinical features, biochemical data and treatment outcome these 60 subjects were classified as either genetic haemochromatosis, nongenetic haemochromatosis or indeterminate. One patient with treated genetic haemochromatosis was excluded from subsequent analysis. RESULTS: Although the serum ferritin (p < 0.002), percentage saturation of transferrin (p < 0.001), histological iron grade (p < 0.0001) were significantly higher in the genetic haemochromatosis than nongenetic haemochromatosis group there was considerable overlap. Similarly for the hepatic iron concentration (HIC) (p < 0.0001) overlap occurred. The hepatic iron index (HIC/age) gave the best separation with only three cases being misclassified. A correlation between the HII and histological iron index (visualised iron score corrected for age) in 15 subjects gave an r value of 0.72. CONCLUSION: Based on this study we feel that in addition to visual grading of iron in liver biopsies, the hepatic iron index is helpful in establishing a diagnosis of genetic haemochromatosis.

Lead exposure during recreational use of small bore rifle ranges.

AIMS: Following detection of symptomatic lead toxicity in two users of an indoor small bore rifle range, we studied users of several similar facilities to determine if significant recreational lead exposure occurred. METHOD: Red cell lead levels were measured at the end of a six month (winter) indoor shooting season and prior to commencement of shooting in the following year. Lead levels in air and dust sampled at one range were also measured. RESULTS: REd cell lead levels were elevated at the end of season (mean 2.64 mumol/L) and lower (mean 1.60 mumol/L) in the preseason samples. The average red cell lead level of the male shooters was 2.4 times normal and is comparable to the levels found in many occupationally exposed groups. Maximum air lead levels were 210 micrograms/m3, more than 2 times the Department of Labour OSH workplace exposure standard TWA of 100 micrograms/m3. Analysis of dust samples showed that dust at this range contained 24% to 36% lead. CONCLUSION: Although the mean time spent shooting was only 70 minutes per week the blood lead levels are similar to those previously reported for full time instructors at pistol ranges. This data confirms that lead exposure in recreational users of indoor small bore rifle ranges is a significant problem.

Increase in selenium status of Christchurch adults associated with deregulation of the wheat market.

AIMS: to determine whether there have been changes in the plasma selenium status of Christchurch adults between 1981 and 1992, and likely causes of any such changes. METHOD: selenium analyses were performed on plasma samples collected during the period. Changes in agricultural practices and importation policies were also examined. RESULTS: mean plasma selenium levels ranged between 46 and 54 micrograms/L (0.59-0.69 mumol/L) until 1987, after which there was a dramatic and sustained increase to between 66 and 70 micrograms/L (0.84-0.88 mumol/L) for 1988-91, with a 1992 mean of 80 micrograms/L (1.01 mumol/L). This increase closely follows the deregulation of the New Zealand wheat market and greater South Island consumption of wheat imported from Australia and the United States. Whereas flour was made from South Island wheat prior to 1988 and contained about 15 micrograms/kg selenium, flour manufactured by Christchurch mills in 1991 contained between 80 and 140 micrograms/kg. CONCLUSIONS: these results imply that as a result of current government policy, the population of Christchurch no longer has particularly low selenium levels. It follows that if there is an association between low selenium and any form of ill health, a declining incidence or severity in this population might be expected.

Industrial blood lead levels in the South Island during 1988 and 1989: trends and follow up patterns.

AIMS: to assess trends in industrial lead exposure and the monitoring programmes in the South Island of New Zealand. METHODS: during the period 1 January 1988 to 31 December 1989, industrial lead exposure was analysed in 1425 workers in at risk occupations and the efficiency of retesting programmes was determined. RESULTS: forty-four percent of these workers had red cell lead levels above 1.9 mumol/L, the top of the reference range for an unexposed population, and 71 individuals had levels exceeding the recommended action limits (males greater than 5.0 mumol/L, and females greater than 3.8 mumol/L). Although most occupational groups showed a small decline in mean red cell lead levels, the pattern of exposure was similar to previous reports. On average, only 43% of exposed workers were retested within the recommended period and 32% of these workers were not retested within 2 years of having a raised blood lead level. CONCLUSIONS: retesting was inefficient but was most reliable when industrial health nurses were employed for monitoring. Not all lead poisoning comes from the traditional lead based industries and significant decreases were found in workers whose primary exposure is to lead from petrol.

Apolipoprotein B-32: a new truncated mutant of human apolipoprotein B capable of forming particles in the low density lipoprotein range.

We have identified a new species of apolipoprotein (apo) B in an individual with heterozygous hypobetalipoproteinemia. The new apo B (apo B-32) is the result of a single point mutation (1450 Gln----Stop) in the apo B gene that prevents full length translation. Apo B-32 is predicted to contain the 1449 amino-terminal amino acids of apo B-100 and is associated with a markedly decreased low density lipoprotein (LDL) cholesterol level. The density distribution of apo B-32 in the plasma lipoproteins makes it unique amongst other truncated apo B species. Normally, apo B-100 is found in both very low density lipoprotein (VLDL) and LDL particles. However, the majority of the apo B-32 protein was found in the high density lipoprotein (HDL) and lipoprotein-deplete (d greater than 1.21 g/ml) fractions, suggesting that it was mainly assembled into abnormally dense lipoprotein particles. A small amount of apo B-32 was also found in the LDL, making it the shortest known apo B variant capable of forming particles in this density range. Apo B-32 was undetected in VLDL. The apo B-32 mutation further defines the minimum length of the apo B protein that is required for the assembly of LDL.

A competitive ELISA for lipoprotein(a).

A competitive ELISA for lipoprotein(a) (Lp(a)) is described. The method uses a commercially available polyclonal anti-Lp(a) antibody and an IgG biotinstreptavidin-horseradish peroxidase detection system. The method is simple and robust with an assay sensitivity of 0.7 ng/well (1.4 micrograms/l). The antibody cross-reactivity was 0.14% against LDL and 0.70% against plasminogen. The coefficients of variation obtained with control sera of 266 and 552 mg/l were: 5.0% and 4.6% (n = 6), respectively for the intraassay; and 10.8% and 9.5% (n = 16), respectively for the interassay. The method showed an excellent correlation with a commercial immunoradiometric assay (IRMA), y (ELISA) = 0.94x (IRMA) - 8, (r = 0.98). A recovery study in which a 200 mg/L standard and four plasma samples were diluted with different proportions of a low plasma sample, gave linear relationships and also confirmed the specificity of the antibody.

Colorimetric measurement of iron in plasma samples anticoagulated with EDTA.

AIMS: To determine if the iron in EDTA anticoagulated plasma samples can be measured by colorimetric assays using Ferrozine. METHODS: Paired samples of serum and EDTA plasma were obtained from 24 patients and analysed by three commercial iron methods. The EDTA plasmas were also analysed using methods modified by the addition of zinc sulphate or with different concentrations of Ferrozine. The iron contamination of EDTA sample tubes was measured by atomic absorption spectroscopy. RESULTS: Two commercial colorimetric iron methods gave results of zero for EDTA plasma samples. A third commercial reagent gave plasma results that were about 30% lower than their corresponding serum samples. Addition of 7 mmol/l zinc sulphate to this reagent system and extending the sample preincubation time to 300 seconds yielded comparable results from paired serum and EDTA plasma samples. Linear regression analysis gave a slope of 0.97 with an intercept of 0.60 mumol/l and R2 = 0.9943. Measurements by atomic absorption spectroscopy showed that this positive intercept was due to contamination of the blood collection tubes with about 90 ng of iron. CONCLUSIONS: Modification of commercial colorimetric iron methods permits the biochemical assessment of iron status and a full blood count from a single EDTA anticoagulated blood sample.

Effect of plasma triglyceride concentrations on the accuracy of immunoturbidimetric assays of apolipoprotein B.

We have studied the influence of triglyceride-rich particles on the analytical bias of apolipoprotein B measurements by various immunoturbidimetric methods. Three commercially available methods grossly overestimate apolipoprotein B in samples with even moderately above-normal triglyceride concentrations. This effect is due to the increased relative reactivity of very-low-density lipoproteins in these reagent systems, and can be eliminated by including Tween 20 (2 g/L) in the reagent buffer. We have developed, and describe, an automated immunoturbidimetric method that allows the accurate determination of apolipoprotein B in the plasma of patients with hypertriglyceridemia.

Hyperlipidemia in stable renal transplant recipients.

Hyperlipidemia is a major risk factor for atherosclerosis and probably contributes to the increased cardiovascular mortality following renal transplantation. We studied the lipid profiles of 62 adults (29 males) with stable renal function (mean plasma creatinine 0.14 mmol/l, SD 0.07), 7 months to 21 years after renal transplantation. Fifteen patients (24%) were above the age- and sex-adjusted 95th percentile for total triglyceride and 10 (16%) for total cholesterol concentrations when compared with a local reference population. The most common lipoprotein abnormalities were type IIa (19%) and type IIb (13%). Multiple regression analysis demonstrated that the use of diuretics and angiotensin-converting enzyme inhibitors were significant factors determining plasma triglyceride concentrations. There were significant bivariate associations between plasma triglyceride concentration and duration since transplantation, plasma creatinine concentration and the use of ciclosporin and diuretics. Duration since transplantation and ciclosporin use were significant factors determining lower plasma cholesterol concentrations. The use of ciclosporin and diuretics was associated with a significantly higher apolipoprotein (apo) B concentration. The cholesterol/HDL cholesterol risk ratio correlated poorly with the apo B/apo A-1 ratio. The value of these ratios as predictors of coronary artery disease need to be established in renal transplant recipients.

Effect of daylight on the reaction of thiols with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid).

The reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) with thiols is sensitive to daylight, in particular to ultraviolet radiation at wavelengths around 325 nm. Exposure to light at the absorbance maximum of the yellow product (the thionitrobenzoate ion) at 410 nm had no effect on the reaction. The light-sensitive species is apparently the DTNB, because a spectral-irradiation experiment showed that the wavelength of light that produced the maximum rate of absorbance change coincided with the peak absorbance of DTNB, and it was well separated from the thionitrobenzoate absorbance peak. Ascorbate is ineffective as a stabilizer and can produce an apparent increase in the rate of DTNB destruction. In a practical example we found the light interference to be severe when hydrolysis of propionylthiocholine by plasma cholinesterase (EC 3.1.1.8) was measured after a 20-min incubation. The apparent cholinesterase activity in clear glass or plastic tubes exposed to diffuse daylight could be decreased to 25% of the value obtained for samples in light-excluded tubes. We recommend the reaction be carried out in artificial room light, with total elimination of daylight, because window glass does not sufficiently attenuate 325-nm wavelength irradiation.