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Long-chain fatty acids (LCFA) play key roles in mammalian cells as sources of energy, structural components and signaling molecules. Given their importance in numerous physiological processes, the roles of LCFAs in health and disease have been extensively investigated. In the majority of studies, correlations are established using a single measurement in plasma or red blood cells (RBCs). Although a few studies have reported on reproducibility of individual fatty acid measurements, the comprehensive analysis of intra-individual LCFA variability has not been performed. Therefore, our goal was to determine intra-individual variability for the 22 most abundant LCFAs in both plasma and RBC samples collected from healthy individuals on a regular diet after overnight fasting. The measurements of LCFAs in RBCs were consistent throughout the course of study reflecting long-term nutritional status. In contrast, the results in plasma showed considerable LCFA intra-individual variability, even between fatty acids of the same type. Plasma intra-individual variability for omega-3 alpha-linolenic and eicosapentaenoic acids in some participants were40% whereas the variability of docosahexaenoic acid was consistently12.8%. Omega-6 linoleic and arachidonic acids also showed low variability in plasma. The results suggest that some LCFAs have less variability and would be more reliable as biomarkers. Reliability of biomarkers can have a profound impact on the research outcomes. Intra-individual variability of LCFAs should be taken into consideration in designing, conducting and interpreting results of clinical studies.

Long-chain fatty acids (LCFAs), including omega-3 and omega-6 fatty acids, play essential roles in health maintenance and outcomes. Insufficient intake or the inability to absorb LCFAs from the diet can cause a number of health problems. Evaluation of fatty acid profiles in plasma, serum or red blood cells (RBCs) is routinely used to monitor patients at risk of developing deficiency. Quantitation of LCFAs in RBCs offers advantages over serum/plasma due to low intra-individual variability. Fatty acid composition in RBCs also reflects long-term dietary intake, providing additional information about the patient's nutritional status. However, the literature does not currently address the impact of pre-analytical factors (conditions of RBC collection, sample handling and short-term storage) on LCFA measurements. This study evaluated the effect of several anticoagulants, interferents, different storage conditions and fasting status on quantitation of the twenty-one most abundant LCFAs in RBCs by gas chromatography negative chemical ionization-mass spectrometry (GC NCI-MS). LCFA results were assessed quantitatively (nmol/mL) or as a percent of total. Most common tube types (containing citrate, sodium heparin or EDTA) were all appropriate for blood collection. Whole blood and lysed RBCs were stable at least 24 h at room temperature and up to 7 days refrigerated. Lysed RBCs were also stable for up to three freeze/thaw cycles. The presence of icterus or lipemia did not affect results. LCFAs concentrations in RBCs did not change ~4 h after high-fat intake when the lipid concentration in circulation reaches a peak, while plasma levels of most fatty acids increased up to 40% in response. In summary, RBCs are a reliable sample type for LCFA quantitation in the clinical laboratory. In contrast to plasma or serum, RBCs isolated from non-fasting, hemolyzed or lipemic whole blood specimens are all acceptable for testing. Therefore, RBCs might be a preferable sample type for evaluation of nutritional status of young pediatric patients and in patients with conditions associated with hemolytic anemia or hyperlipidemia.

The most common causative mutation of Friedreich ataxia (FRDA) is the unstable hyperexpansion of an intronic GAA triplet repeat that impairs frataxin transcription. Using real time quantitative PCR, we showed that FRDA patients had residual levels of frataxin mRNA ranging between 13% and 30% and that FRDA carriers had about 40% of that of controls. Asymptomatic carriers also showed reduced frataxin mRNA levels. We found an inverse correlation between the number of GAA repeats and frataxin mRNA levels. Real-time quantitative PCR may represent an alternative assay for FRDA molecular diagnosis.

3 Diabetes mellitus or carbohydrate intolerance is frequently described. 1 The age of onset is variable. The disease usually comes at puberty but several cases have become symptomatic after 40 years. 4 The mole- cular defect that occurs in Friedreich's ataxia is the trinucleotide GAA expansion in the first intron of the X25 gene. This gene encodes for a 210 aa mitochondrial protein called frataxin. 5 Levels of frataxin mRNA and protein are severely reduced as a result of this expansion. 5 Although the exact physiological function of frataxin is not known, its involvement in iron-sulphur (Fe-S) cluster biogenesis 6 has been suggested. The Friedreich's ataxia phenotype shows a variable expression with respect to the age of onset and the presence of some signs or symptoms. Disease duration has been proposed to influence dysarthria, decreased vibration sense, optic atrophy, and diabetes. 7 Some Friedreich's ataxia phenotype features are strongly correlated with GAA expan- sion size. An inverse relationship has been demonstrated between GAA repeat size and the age of onset. 78 Friedreich's ataxia with retained reflexes or absence of cardiomyopathy was associated with shorter expansions. In a previous report, we demonstrated that GAA size accounts for about 70% of the onset age variance, 8

Friedreich’s ataxia (FRDA) is the most common hereditary ataxia, with a birth incidence of 1 in 25,000. The most frequent cause, present in approximately 98% of FRDA chromosomes, is an abnormal GAA expansion in the first intron of the X25 gene. The presence of this expansion inhibits gene expression, resulting in decreased levels of the encoded protein frataxin. X25 missense, nonsense, and splice site mutations have been identified in the remaining FRDA chromosomes.1 Frataxin is located in mitochondria. The localization has been proved by immunocytofluorescence with monoclonal antibodies, great frataxin enrichment in the mitochondrial pellet obtained by differential centrifugation, and immunoelectron microscopy.2 Even though the exact function of frataxin remains unknown, it seems to be important in mitochondrial iron handling. Deletion of the yeast frataxin homologue, YFH1 , results in a 10-fold increased iron in mitochondria, a reduction in mitochondrial DNA, reduced respiration, increased sensitivity to oxidative stress, and decreased activities of iron-sulfur protein levels. Cytosolic iron is decreased with upregulation of high-affinity iron uptake proteins.3,4⇓ Evidences of iron accumulation in humans are …