Brian M. Gilfix

Genetic polymorphisms in methylenetetrahydrofolate reductase and methionine synthase, folate levels in red blood cells, and risk of neural tube defects

Folic acid administration to women in the periconceptional period reduces the occurrence of neural tube defects (NTDs) in their offspring. A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR), 677C-->T, is the first genetic risk factor for NTDs in man identified at the molecular level. The gene encoding another folate-dependent enzyme, methionine synthase (MTR), has recently been cloned and a common variant, 2756A-->G, has been identified. We assessed genotypes and folate status in 56 patients with spina bifida, 62 mothers of patients, 97 children without NTDs (controls), and 90 mothers of controls, to determine the impact of these factors on NTD risk. Twenty percent of cases and 18% of case mothers were homozygous for the MTHFR polymorphism, compared to 11% of controls and 11% of control mothers, indicating that the mutant genotype conferred an increased risk for NTDs. The risk was further increased if both mother and child had this genotype. The MTR polymorphism was associated with a decreased O.R. (O.R.); none of the cases and only 10% of controls were homozygous for this variant. Red blood cell (RBC) folate was lower in cases and in case mothers, compared to their respective controls. Having a RBC folate in the lowest quartile of the control distribution was associated with an O.R. of 2.56 (95% CI 1.28-5.13) for being a case and of 3.05 (95% CI 1.54-6.03) for being a case mother. The combination of homozygous mutant MTHFR genotype and RBC folate in the lowest quartile conferred an O.R. for being a NTD case of 13.43 (CI 2.49-72.33) and an O.R. for having a child with NTD of 3.28 (CI 0.84-12.85). We propose that the genetic-nutrient interaction--MTHFR polymorphism and low folate status--is associated with a greater risk for NTDs than either variable alone.

Treatment of Vitamin B12 Deficiency after Gastric Surgery for Severe Obesity

Background: Vitamin B12 deficiency after gastric surgery for obesity is due to a failure of separation of vitamin B12 from protein foodstuffs and to a failure of absorption of crystalline vitamin B12 in the presence of intrinsic factor. The purpose of this study was to determine which of four oral doses of crystalline vitamin B12 was most effective in treating vitamin B12 deficiency in 102 patients. Methods and Results: At time of entry into the study, the patients had a serum vitamin B12 < 100 pmol L −1, were 29.9 ± 21.7 months post-op, were 37 ± 8 years old and had a body mass index of 30 ± 6 kg m−2. Eight (8%) had had a vertical banded gastroplasty and 94 (92%) a gastric bypass. For the first 3 months all patients received 350 μg per day of crystalline vitamin B12 and all increased their serum vitamin B12 levels to over 100 pmol L−1. The patients were then assigned to receive for a further 3 month period one of four oral doses of crystalline vitamin B12-100 μg, 250 μg, 350 μg and 600 μg. Serum vitamin B12 levels were greater than 150 pmol L−1 after 6 months in 83.3% of patients who received 100 μg; 92.3% of patients who received 250 μg; 94.7% after 350 μg and 95.2% after 600 μg (p%0.525). Conclusion: At least 350 μg per day is the appropriate oral dose of crystalline vitamin B12 after gastric surgery for obesity to correct low serum vitamin B12 levels in 95% of patients.

Review of the effect of intravenous lipid emulsion on laboratory analyses

Abstract Context Although the clinical use of intravenous lipid emulsion therapy for the treatment of lipophilic drug toxicity is increasing, the focus of most publications is on outcome in laboratory animals or in patients. An unintended consequence of intravenous lipid emulsion is the creation of extremely lipemic blood, which may interfere with the laboratory analysis or interpretation of common analytes. Objective The American Academy of Clinical Toxicology has established a lipid emulsion workgroup to review the evidence and produce recommendations on the use of this novel therapy for drug toxicity. The aim of this subgroup is to review the available evidence regarding the effect of intravenous lipid emulsion on common laboratory testing, which often forms the basis of the appraisal of the balance between benefits and potential adverse events. Methods We performed a comprehensive review of the literature. Relevant articles were determined based upon a predefined methodology. Package inserts of manufacturers’ assays were collected. Article inclusion required that the article met predefined inclusion criteria with the agreement of at least two members of the subgroup. Results We included thirty-six articles in the final analysis. Evaluation of the reviewed analytes revealed heterogeneity with regards to the assessment of the effect of intravenous lipid emulsion in terms of consistency and magnitude of effect across the different analytic platforms. Conclusions The measurements of a number of common analytes can be markedly affected by the lipemia produced by lipid emulsions such that they cannot always be interpreted in the way that most physicians use this information in typical clinical situations. In fact, a lack of appreciation of this effect may lead to unintentional treatment errors. Because the effect of the lipemia produced is dependent on the reagents and laboratory platform used, it would be useful for all future reports to clearly document sample handling, reagents and laboratory platform used, as well as any procedures employed to reduce the lipid content.

Analytical interferences resulting from intravenous lipid emulsion

Context. Lipid resuscitation therapy using intravenous lipid emulsion (IVLE) for drug overdoses has gained widespread use. However, there is little information regarding its adverse effects. Objectives. We performed lipemic interference studies on typical automated platforms to investigate the potential of lipid resuscitation therapy to interfere with the reliability and turnaround time of analytes that would be of interest in acute intoxications. We also tested methods to minimize interferences. Materials and methods. Serum pools were supplemented with increasing concentrations of Intralipid-20%® (0–30%). Analyses were performed on Beckman-Coulter DXC800 and DXI and Roche Modular-P. Analytes demonstrating significant interference were re-measured after centrifugation (14 000 × g for 10 minutes). Results. Triglyceride and glycerol-blanked triglyceride concentrations were similar in IVLE-free samples. However, with addition of IVLE, concentrations were markedly different (139 vs. 76 mmol/L). There was no appreciable interference on the troponin-I, sodium, potassium, chloride, calcium, bicarbonate or urea assays. Albumin and magnesium assays demonstrated significant interference. Amylase, lipase, phosphate, creatinine, total protein, ALT, CK and bilirubin became unmeasurable in IVLE-supplemented samples. Whereas glucose measurement by potentiometry was free of interference, colorimetric methodology was error prone. Centrifugation removed > 90% of glycerol-blanked triglyceride (max = 5.8 mmol/L), dramatically reducing lipid interferences. Discussion. IVLE results in appreciable analytical interferences at concentrations demonstrated in lipid resuscitation therapy. Of particular concern is the marked interference on glucose and magnesium, which may result in unsuccessful and potentially harmful interventions. Major implications for patient care include reporting of incorrect results and delays in the reporting of time-sensitive results. Whenever possible, blood samples should be collected prior to initiating lipid therapy. Interferences can be minimized by brief centrifugation at relatively low speeds on equipment readily available in most core labs.

Seven novel mutations in mut methylmalonic aciduria

Methylmalonic aciduria (MMA) is an autosomal recessive inborn error of metabolism that results from functional defects in methylmalonyl CoA mutase (MCM), a nuclear‐encoded, mitochondrial enzyme that uses the vitamin B12 derivative, adenosylcobalamin (AdoCbl) as a cofactor. To date, 23 mutations have been identified at the MUT locus on the short arm of chromosome 6, causing the mut forms of MMA (mut complementation group; mut MMA, McKusick #251000). We now report seven novel mutations. Three were found in mut0 patients: R228Q (c759G→A) was found as a heterozygous change; G312V (c1011G→T) and 346delL (c1112delCTT) were both found as homozygous changes. Four mutations were found in mut– patients: A191E (c648C→A) and V633G (c1974T→G) were found in the same patient; 684insL (c2128insCTC) and L685R (c2130T→G) were both found as homozygous changes. The recent modelling of the human methylmalonyl CoA mutase allowed for an interpretation of the identified mutations. Hum Mutat 11:270–274, 1998.

Pilot Study Examining the Frequency of Several Gene Polymorphisms in a Morbidly Obese Population

Background: Obesity is a growing problem and is associated with numerous medical conditions. Several polymorphisms have been associated with lipid metabolism and obesity: PPARγ2-Pro115Gln, PPARγ2-Pro12Ala, β3AR-Trp64Arg and SR-BI IVS5 C>T. We examined the frequency of these polymorphisms in patients with a BMI>40 and compared them to individuals with a BMI<30. Our hypothesis was that these polymorphisms would occur more frequently in the obese population. Methods: This case-control study examined 126 individuals with a BMI>40 in the McGill University Health Centre Bariatric Surgery Program and 102 individuals (controls) with a BMI<30 attending a Lipid Clinic. DNA was extracted from whole blood by standard techniques. The polymorphisms were determined by polymerase chain reaction (PCR) and restriction genotyping. Results: A significant difference between controls and the morbidly obese group was observed for 2 of 4 polymorphisms.The carrier frequency for PPARγ2-Pro12Ala was 24.8% in the obese group and 12.9% in controls (odds ratio = 2.2; 95%CI = 1.1-4.4; P=0.02). The carrier frequency for SR-BI IVS5 C>T was 22.8% in obese individuals versus 8.1% in controls (odds ratio = 3.5; 95%CI = 1.6-7.7; P=0.002). There were no differences in frequency of diabetes in both obese (13/104) and control (9/126) groups (odds ratio = 1.86, 95%CI= 0.76<O.R.<4.54, P=0.184). Conclusions: These results underscore the rela tionship between gene polymorphisms and obesity. Obese individuals may differ from non-obese individuals in the gene polymorphisms associated with metabolic control.

Impact of transfusion of mediastinal shed blood on serum levels of cardiac enzymes

BACKGROUND Infusion of shed mediastinal blood using an autotransfusion system is a widely applied technique of blood conservation in cardiac surgery. Serial determinations of serum creatine kinase (CK), its MB isoenzyme (CK-MB), and lactate hydrogenase (LDH) levels have been used to monitor perioperative myocardial injury. We investigated the impact of postoperative autotransfused blood infusion on serum levels of these enzymes. METHODS We performed a retrospective analysis of postoperative serum CK, CK-MB, and LDH levels of 300 patients who had elective uncomplicated aortocoronary bypass grafting. Shed mediastinal blood samples from 26 patients were analyzed for CK, CK-MB (enzymatic activity and mass), and LDH levels before infusion. RESULTS High postoperative serum levels of CK and LDH were observed after infusion of autotransfused blood. Shed mediastinal blood contained extremely high levels of these enzymes, particularly from patients who had internal mammary artery dissection. There was a strong correlation (r = 0.96) between measured CK-MB enzyme activities and those calculated from the CK-MB mass units. CONCLUSIONS Infusion of autotransfused blood containing high concentrations of CK and LDH results in elevated serum levels of these enzymes. Hemolysis, frequently present in shed blood, does not interfere with the routine biochemical assays for CK and CK-MB enzyme activities. Caution should be taken when postoperative cardiac enzyme levels are used to determine myocardial injury after aortocoronary bypass grafting if autotransfusion is used as a method of blood conservation.

Methodology for AACT evidence-based recommendations on the use of intravenous lipid emulsion therapy in poisoning

Abstract Intravenous lipid emulsion (ILE) therapy is a novel treatment that was discovered in the last decade. Despite unclear understanding of its mechanisms of action, numerous and diverse publications attested to its clinical use. However, current evidence supporting its use is unclear and recommendations are inconsistent. To assist clinicians in decision-making, the American Academy of Clinical Toxicology created a workgroup composed of international experts from various clinical specialties, which includes representatives of major clinical toxicology associations. Rigorous methodology using the Appraisal of Guidelines for Research and Evaluation or AGREE II instrument was developed to provide a framework for the systematic reviews for this project and to formulate evidence-based recommendations on the use of ILE in poisoning. Systematic reviews on the efficacy of ILE in local anesthetic toxicity and non-local anesthetic poisonings as well as adverse effects of ILE are planned. A comprehensive review of lipid analytical interferences and a survey of ILE costs will be developed. The evidence will be appraised using the GRADE system. A thorough and transparent process for consensus statements will be performed to provide recommendations, using a modified Delphi method with two rounds of voting. This process will allow for the production of useful practice recommendations for this therapy.

Genetic and molecular control of folate-homocysteine metabolism in mutant mice

Abstract. Hyperhomocysteinemia adversely affects fundamental aspects of fetal development, adulthood, and aging, but the role of elevated homocysteine levels in these birth defects and adult diseases remains unclear. Mouse models are valuable for investigating the causes and consequences of hyperhomocysteinemia. We used a phenotype-based approach to identify mouse mutants for studying the relation between single gene mutations, homocysteine levels as a measure of the status of homocysteine metabolism, and gene expression profiles as a way to assess the impact of protein deficiency in mutant mice on steady-state transcription levels of genes in the folate-homocysteine pathways. These mutants were selected based on their propensity to produce phenotypes that are reminiscent of those associated with anomalies in folate-homocysteine metabolism in humans. We report identification of new, single-gene mouse models of homocysteinemia and characterization of their molecular and physiological impact on folate-homocysteine metabolism. Mutations in several genes involved in the hedgehog and WNT signal transduction pathways, as well as a gene involved in lipid metabolism, resulted in elevated homocysteine levels and altered expression profiles of folate-homocysteine metabolism genes. These results begin to unravel the complex relations between elevation of a single amino acid in the blood and the diverse birth defects and adult diseases associated with hyperhomocysteinemia.

Spina bifida, folate metabolism, and dietary folate intake in a Northern Canadian aboriginal population

Objectives. Inhabitants of the subarctic region of the Eastern James Bay of Northern Quebec consume a diet low in folate. This is largely secondary to poor access to plant-foods and a preferred diet high in meat, fowl, and fish as in many other northern populations. Furthermore, there is a high frequency of spina bifida in the Cree of the region. It was hypothesized that genetically altered folate metabolism as well as low folate intake contributes to the high frequency of spina bifida. Methods: A casecontrol study evaluating folate metabolism and the common 677C-T polymorphism of the gene for methylenetetrahydrofolate reductase (MTHFR) in mothers of children with spina bifida, and controls (n=23) of Cree descent from the Eastern James Bay region. These results were compared to a similar Montreal cohort (n=152) who were not of First Nations descent. Dietary intake of folate of 219 women of the Eastern James Bay region was also determined. Results: No Cree mothers of children with spina bifida were homozygous for the 677C-T polymorphism of MTHFR. Although serum cobalamin was significantly higher in Cree mothers, RBC folate was significantly lower than in the Montreal cohort. In addition, plasma homocysteine was significantly lower in the Cree. Dietary intake of folate of women in the same region was substantially lower (100 µg/day) than widely recommended daily intakes. Conclusions. In this remote Canadian aboriginal community there is no evidence of altered folate metabolism in the mothers of children with spina bifida. Nonetheless, it remains essential that culturally appropriate public health efforts be continued to increase the intake of folic acid in the hope of reducing the high frequency of spina bifida in this population.