Plasma elemental responses to red meat ingestion in healthy young males and the effect of cooking method.

PURPOSE: Elemental deficiencies are highly prevalent and have a significant impact on health. However, clinical monitoring of plasma elemental responses to foods remains largely unexplored. Data from in vitro studies show that red meat (beef) is a highly bioavailable source of several key elements, but cooking method may influence this bioavailability. We therefore studied the postprandial responses to beef steak, and the effects of two different cooking methods, in healthy young males. METHODS: In a randomized cross-over controlled trial, healthy males (n = 12, 18-25 years) were fed a breakfast of beef steak (270 ± 20 g) in which the meat was either pan-fried (PF) or sous-vide (SV) cooked. Baseline and postprandial blood samples were collected and the plasma concentrations of 15 elements measured by inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: Concentrations of Fe and Zn changed after meal ingestion, with plasma Fe increasing (p < 0.001) and plasma Zn decreasing (p < 0.05) in response to both cooking methods. The only potential treatment effect was seen for Zn, where the postprandial area under the curve was lower in response to the SV meal (2965 ± 357) compared to the PF meal (3190 ± 310; p < 0.05). CONCLUSIONS: This multi-element approach demonstrated postprandial responsiveness to a steak meal, and an effect of the cooking method used. This suggests the method would provide insight in future elemental metabolic studies to evaluate responses to meat-based meals, including longer-term interventions in more specifically defined cohorts to clearly establish the role of red meat as an important source of elements.

Relative quantification of albumin and fibrinogen modifications by liquid chromatography tandem mass spectrometry in the diagnosis and monitoring of acute pancreatitis.

The increasing availability of liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories provides the opportunity to replace or complement present underperforming immuno- and chemometric assays. Amylase and lipase show limited specificity and sensitivity for pancreatic inflammation and lack the capacity of monitoring the disease due to their short half-lives. Previous findings suggested that cleavage products of the pancreatic enzyme carboxypeptidase A could be a more suitable indicator for defining and classifying pancreatic inflammation. The plasma proteins albumin and ß-fibrinogen were digested with trypsin and truncated forms (des-Leu-albumin, and des-Gln-ß-fibrinogen) quantified against their non-truncated forms by LC-MS/MS. Four hundred fifty eight samples from 83 patients were used to evaluate the novel method and affirm its suitability for detecting acute pancreatitis. A robust, selective, precise and accurate LC-MS/MS method was set up to measure the proportion of truncated proteins. Reference ranges for the proportion of the truncated albumin and ß-fibrinogen were from 2% to 9% and 3% to 25%, respectively. Acute pancreatitis patients had values above these ranges and were distinctly separated from reference control individuals. The longer circulating half-lives of albumin and fibrinogen compared to pancreatic enzymes themselves provide the potential to diagnose pancreatitis more specifically over a longer time period, to monitor the course of the disease, and to track recurrent complications. The wide range of the proportion and the differential half-life of both truncated proteins could also be used for assessing the severity of pancreatitis.

Laboratory medicine best practice guideline: vitamins a, e and the carotenoids in blood.

Despite apparent method similarities between laboratories there appear to be confounding factors inhibiting uniform reporting and standardisation of vitamin assays. The Australasian Association of Clinical Biochemists (AACB) Vitamins Working Party, in conjunction with The Royal College of Pathologists of Australasia Quality Assurance Programs, has formulated a guideline to improve performance, reproducibility and accuracy of fat-soluble vitamin results. The aim of the guideline is to identify critical pre-analytical, analytical and post-analytical components of the analysis of vitamins A, E and carotenoids in blood to promote best practice and harmonisation. This best practice guideline has been developed with reference to the Centers for Disease Control and Prevention (CDC) "Laboratory Medicine Best Practices: Developing an Evidence-Based Review and Evaluation Process". The CDC document cites an evaluation framework for generating best practice recommendations that are specific to laboratory medicine. These 50 recommendations proposed herein, were generated from a comprehensive literature search and the extensive combined experience of the AACB Vitamins Working Party members. They were formulated based on comparison between an impact assessment rating and strength of evidence and were classified as either: (1) strongly recommend, (2) recommend, (3) no recommendation for or against, or (4) recommend against. These best practice recommendations represent the consensus views, in association with peer reviewed evidence of the AACB Vitamins Working Party, towards best practice for the collection, analysis and interpretation of vitamins A, E and carotenoids in blood.

Falsely elevated plasma selenium due to gadolinium contrast interference: a novel solution to a preanalytical problem.

BACKGROUND: Trace elements are commonly measured by inductively coupled mass spectrometry (ICP-MS). A 30-year-old man had a plasma selenium (Se) concentration on ICP-MS of 66 µmol/L (reference interval 0.45-1.40), a potentially lethal level, despite no history of Se exposure or toxicity symptoms. He had earlier undergone magnetic resonance imaging with a gadolinium (Gd) contrast agent, which is known to interfere with Se on ICP-MS. We aimed to adjust our method by monitoring a second Se isotope that is unaffected by Gd to detect this preanalytical interference. METHODS: Plasma samples referred for trace metal testing had Se measured on ICP-MS (monitoring (78)Se), which we modified to also monitor a second isotope ((82)Se). The modified method was then applied to a specimen with known Gd contamination. RESULTS: Plasma Se results (n = 41) derived from monitoring the two different Se isotopes were similar with a good correlation (R (2 )= 0.991) over a range of 0.23-2.21 µmol/L. On repeat analysis, our patient had a Se concentration of 65 µmol/L using the (78)Se isotope but only 1.43 µmol/L using (82)Se. CONCLUSION: To avoid reporting a falsely elevated plasma Se result, we suggest that Se analysis by ICP-MS should include a second Se isotope for monitoring, that is not subject to Gd interference.

Vitamin B1 and B6 method harmonization: comparison of performance between laboratories enrolled in the RCPA Quality Assurance Program.

OBJECTIVES: The RCPA Quality Assurance Program (RCPA QAP) offers monthly proficiency testing for vitamins A, B1, B6, ß-carotene, C and E to laboratories worldwide. A review of the results submitted for the whole blood vitamin B1/B6 sub-program revealed a wide dispersion. Here we describe the results of a methodology survey for vitamins B1 and B6. DESIGN AND METHODS: A questionnaire was sent to thirteen laboratories. Eleven laboratories were returning QAP results for vitamin B1 (thiamine diphosphate) and five were returning results for vitamin B6 (pyridoxal-5-phosphate). RESULTS: All nine respondents provided a clinical service for vitamins B1 and B6. HPLC with fluorescence detection was the most common method principle. For vitamin B1, six respondents used a commercial assay whilst three used in-house methods; whole blood was the matrix for all. For vitamin B6, five respondents used commercial assays and four used in-house assays. The choice of matrix for vitamin B6 varied with three respondents using whole blood and five using plasma for analysis. Sample preparation incorporated protein precipitation and derivatization steps. An internal standard was employed in sample preparation by only one survey respondent. CONCLUSIONS: The immediate result of this survey was the incorporation of plasma vitamin B6 into the RCPA QAP vitamin program. The absence of an internal standard in current vitamin B1 and B6 assays is a likely contributor to the wide dispersion of results seen in this program. We recommend kit manufacturers and laboratories investigate the inclusion of internal standards to correct the variability that may occur during processing.

A mass-spectroscopic method for measuring des-Leu albumin--a novel marker for chronic pancreatitis.

OBJECTIVES: Chronic pancreatitis is a progressive inflammatory disease leading to pancreatic insufficiency. The diagnosis of chronic pancreatitis is challenging, especially in early disease and the current tests have low sensitivity, may be invasive or have limited availability. We previously identified a truncated form of albumin lacking the C-terminal leucine, des-Leu albumin, which was present at high concentration in pancreatitis. We have developed a liquid-chromatography tandem-mass spectrometry (LC-MS/MS) method for measuring this peptide and make some preliminary observations on patient samples. METHODS: Serum samples from patients with established pancreatitis and controls were obtained. Diluted serum samples or prepared standards were digested with trypsin. Aliquots of the digest were separated on a reversed-phase column using water:acetonitrile:formic acid mobile-phase with tandem-mass spectrometry detection. Percentage composition of des-Leu albumin was determined from a response curve. RESULTS: The C-terminal peptide, LVAASQAALG- of des-Leu albumin was identified by m/z 901→725, wild type albumin by m/z 1014→825. Additional fragments were monitored as internal reference for digestion and sample integrity. Inter-assay imprecision was estimated at 10%. The percentage composition of des-Leu albumin segregated with the diagnosis of established pancreatitis with median levels of des-Leu albumin of 68% in patients compared to 5% in controls. CONCLUSIONS: Des-Leu albumin is a promising novel biomarker for chronic pancreatitis. It allowed clear discrimination of patients with pancreatitis from controls and its long half-life may facilitate monitoring of disease activity. The method described could readily be undertaken in modern clinical chemistry laboratories and will form the basis for further study.

Thiopurine methyltransferase and thiopurine metabolite testing in patients with inflammatory bowel disease who are taking thiopurine drugs.

Thiopurine methyltransferase genotyping and thiopurine metabolite testing has been established as an adjunct to monitoring patients taking thiopurine drugs. This special report describes the clinical implications for this type of testing for patients with inflammatory bowel disease who are taking thiopurine drugs. A total of 10% of patients were found to be intermediate metabolizers and the mean dosage (in mg/kg equivalent) was lower in intermediate metabolizers than extensive metabolizers. The metabolite levels did not correlate with scores measuring clinical severity but levels of 6-methylmercaptopurine were related to the dosage of the drugs. Despite considerable study of thiopurine methyltransferase testing in the literature, it is still not widely used in many geographical areas. This study adds to the evidence about using such testing as well as expanding the role of simultaneously measuring thiopurine metabolites. Further work is planned to evaluate the uptake when such testing becomes available locally as a clinical service.

Folate and vitamin B12 status of women of reproductive age living in Hanoi City and Hai Duong Province of Vietnam.

OBJECTIVES: To assess the folate and vitamin B12 status of a group of Vietnamese women of reproductive age and to estimate the rate of neural tube defects (NTD) based on red blood cell (RBC) folate concentrations. DESIGN AND SUBJECTS: A representative sample of non-pregnant women (15-49 years) living in Hanoi City (n 244) and Hai Duong Province (n 245). MEASURES: RBC folate, plasma vitamin B12 and plasma holo-transcobalamin (holoTC), a sensitive indicator of vitamin B12 status. RESULTS: Mean (95% CI) concentrations of RBC folate, plasma B12 and plasma holoTC were 856 (837, 876) nmol/l, 494 (475, 513) pmol/l and 78 (74, 82) pmol/l, respectively. Only 3% and 4% of women had plasma B12 and holoTC concentrations indicative of deficiency. No woman had an RBC folate concentration indicative of deficiency (<317 nmol/l). Only 47% of women had an RBC folate concentration > or = 905 nmol/l. Accordingly, we predict the NTD rate in these regions of Vietnam to be 14.7 (14.2, 15.1) per 10,000 pregnancies. CONCLUSION: There was no evidence of folate and vitamin B12 deficiency among this population of Vietnamese women. However, suboptimal folate status may be placing three out of five women at increased risk of NTD. Reductions in NTD rates are still possible and women would benefit from additional folic acid during the periconceptional period from either supplements or fortified foods.

Lead poisoning due to ingestion of Indian herbal remedies.

A case of lead poisoning is presented. The patient had recently returned to New Zealand from the Indian subcontinent. This prompted a search that identified lead contamination of ingested medicinal products that had been prescribed in India. There have been several case reports of lead toxicity due to contamination of Indian herbal medicines, though none, to our knowledge, previously reported from New Zealand.

Effect of volcanic gas exposure on urine, blood, and serum chemistry.

AIMS: This pilot study tested the hypothesis that aluminium (Al), rubidium (Rb), arsenic (As), lead (Pb), mercury (Hg), fluorine (F), and chlorine (Cl), which are all known to be present in volcanic emissions, may be useful biological markers for occupational gas exposure in volcanologists. METHODS: Ten human subjects were exposed to fumarole gases on White Island, New Zealand, for approximately 20 minutes. Sulphur dioxide (SO2) exposure was recorded by personal monitoring tubes. Pre- and post-exposure urine, blood and serum samples (collected using standard protocols) were analysed in the pathology laboratory for trace element and halogen content. RESULTS: Average personal exposure was measured at <75 ppm SO2 and calculated at approximately 25 ppm HCl, approximately 8 ppm hydrogen fluoride (HF), approximately 1 ppm Al, approximately 0.1 ppb Rb and approximately 4 ppb Pb. These concentrations almost certainly exceed those usually found in occupational exposure settings. Advanced levels of urinary Al and Rb were found following gas exposure and were statistically significant in the population at p<0.005 and p<0.001, respectively. The other chemical elements that were analysed (urinary Cl, F, and Hg; blood Pb, and serum Al) did not show such patterns. CONCLUSIONS: It is possible that urinary Al and Rb may be useful markers for exposure, a hypothesis which should be followed up in future work.

Measurement of thiopurine methyl transferase activity guides dose-initiation and prevents toxicity from azathioprine.

AIM: To establish an assay service for thiopurine methyl transferase (TPMT) activity in order to facilitate dose initiation of thiopurine drug therapy and to define appropriate reference intervals and optimal cut-offs for the New Zealand population. METHODS: 407 patients underwent radio-enzymatic assay testing of TPMT activity prior to initiation of thiopurine drug therapy. Those with low activity also underwent genotyping for the abnormal *2, *3A, and *3C alleles. RESULTS: A trimodal distribution of enzyme activity was seen consistent with the known polymorphic genetics for this enzyme. Three cases of homozygous deficiency were identified. The 'normal' range is 9.3 to 17.6 units/ml red blood cells (RBCs), but many heterozygotes have activity above the lower limit of his range. TPMT activity above 10.7 units/ml RBC identifies a normal genotype with 100% probability. CONCLUSION: The normal range for TPMT has been established. The measurement of TPMT activity helps to guide dose initiation and may prevent toxicity from azathioprine.