Multiplexing Iduronate-2-Sulphatase (MPS-II) into a 7-Plex Lysosomal Storage Disorder MS/MS Assay Using Cold-Induced Phase Separation.

Mucopolysaccharidosis type II (MPS-II, Hunter syndrome, OMIM:30990) is a lysosomal storage disorder (LSD) that results in iduronate 2-sulphatase (I2S) enzyme deficiency. MPS-II was added to the Recommended Uniform Screening Panel (RUSP) in August 2022; thus, there is an increased demand for multiplexing I2S into existing LSD screening assays. After incubation with LSD synthetic substrates, extracts are cleaned using liquid-liquid extraction with ethyl acetate or protein precipitation using acetonitrile (ACN). We investigated cold-induced water ACN phase separation (CIPS) to improve the combination of 6-plex and I2S extracts to create a 7-plex assay, and compared it to room temperature ACN and ethyl acetate liquid-liquid extraction. The extracts were dried and resuspended in the mobile phase, and then analyzed using an optimized 1.9 min injection-to-injection liquid chromatography method coupled with tandem mass spectrometry (LC-MS/MS). The combination of ACN and CIPS improved the detection for I2S products without significant detriment to other analytes, which is attributable to a more complete coagulation and separation of heme, proteins, and extracted residual salts. Using CIPS for sample cleanup in dried blood spots (DBS) appears to represent a promising and straightforward way of achieving cleaner sample extracts in a new 7-plex LSD screening panel.

Multiplexing Homocysteine into First-Tier Newborn Screening Mass Spectrometry Assays Using Selective Thiol Derivatization.

BACKGROUND: Classical homocystinuria (HCU) results from deficient cystathionine ß-synthase activity, causing elevated levels of Met and homocysteine (Hcy). Newborn screening (NBS) aims to identify HCU in pre-symptomatic newborns by assessing Met concentrations in first-tier screening. However, unlike Hcy, Met testing leads to a high number of false-positive and -negative results. Therefore, screening for Hcy directly in first-tier screening would be a better biomarker for use in NBS. METHODS: Dried blood spot (DBS) quality control and residual clinical specimens were used in analyses. Several reducing and maleimide reagents were investigated to aid in quantification of total Hcy (tHcy). The assay which was developed and validated was performed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS). RESULTS: Interferents of tHcy measurement were identified, so selective derivatization of Hcy was employed. Using N-ethylmaleimide (NEM) to selectively derivatize Hcy allowed interferent-free quantification of tHcy by FIA-MS/MS in first-tier NBS. The combination of tris(2-carboxyethyl)phosphine (TCEP) and NEM yielded significantly less matrix effects compared to dithiothreitol (DTT) and NEM. Analysis of clinical specimens demonstrated that the method could distinguish between HCU-positive, presumptive normal newborns, and newborns receiving total parenteral nutrition. CONCLUSIONS: Here we present the first known validated method capable of screening tHcy in DBS during FIA-MS/S first-tier NBS.

Development of a Universal Second-Tier Newborn Screening LC-MS/MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids.

First-tier MS-based newborn screening by flow injection analysis can have high presumptive positive rates, often due to isomeric/isobaric compounds or poor biomarker specificity. These presumptive positive samples can be analyzed by second-tier screening assays employing separations such as liquid chromatography-mass spectrometry (LC-MS/MS), which increases test specificity and drastically reduces false positive referrals. The ability to screen for multiple disorders in a single multiplexed test simplifies workflows and maximizes public health laboratories' resources. In this study, we developed and validated a highly multiplexed second-tier method for dried blood spots using a hydrophilic interaction liquid chromatography (HILIC) column coupled to an MS/MS system. The LC-MS/MS method was capable of simultaneously detecting second-tier biomarkers for maple syrup urine disease, homocystinuria, methylmalonic acidemia, propionic acidemia, glutaric acidemia type 1, glutaric acidemia type 2, guanidinoacetate methyltransferase deficiency, short-chain acyl-CoA dehydrogenase deficiency, adrenoleukodystrophy, and Pompe disease.

High resolution mass spectrometry newborn screening applications for quantitative analysis of amino acids and acylcarnitines from dried blood spots.

Amino acid and acylcarnitine first-tier newborn screening typically employs derivatized or non-derivatized sample preparation methods followed by FIA coupled to triple quadrupole (TQ) MS/MS. The low resolving power of TQ instruments results in difficulties distinguishing nominal isobaric metabolites, especially those with identical quantifying product ions such as malonylcarnitine (C3DC) and 4-hydroxybutylcarnitine (C4OH). Twenty-eight amino acids and acylcarnitines extracted from dried blood spots (DBS) were analyzed by direct injection (DI)-HRMS on a Q-Exactive Plus across available mass resolving powers in SIM, in PRM at 17,000 full width at half maximum (FWHM), and a developed SIM/PRM hybrid MS method. Most notably, quantitation of C3DC and C4OH was successful by HRMS in non-derivatized samples, thus, potentially eliminating sample derivatization requirements. Quantitation differed between SIM and PRM acquired data for several metabolites, and it was determined these quantitative differences were due to collision energy differences or kinetic isotope effects between the unlabeled metabolites and the corresponding labeled isotopologue internal standards. Overall quantitative data acquired by HRMS were similar to data acquired on TQ MS/MS platform. A proof-of-concept hybrid DI-HRMS and SIM/PRM/FullScan method was developed demonstrating the ability to hybridize targeted newborn screening with metabolomic screening.

Development of dried blood spot quality control materials for adenosine deaminase severe combined immunodeficiency and LC-MS/MS method for their characterization.

Adenosine deaminase severe combined immunodeficiency (ADA-SCID) is an autosomal recessive disorder in which a lack of ADA enzyme prevents the maturation of T- and B-cells; early intervention is crucial for restoring immune function in affected neonates. ADA is responsible for purine metabolism and-in its absence-adenosine, deoxyadenosine, and S-adenosylhomocysteine build up and can be detected in the blood. Preparing dried blood spot (DBS) quality control (QC) materials for these analytes is challenging because enrichments are quickly metabolized by the endogenous ADA in normal donor blood. Adding an inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), has been previously reported to minimize enzyme activity, although this adds additional cost and complexity. We describe an alternative method using unnatural L-enantiomer nucleosides (L-adenosine and 2'-deoxy-L-adenosine) which eliminates the need for enzyme inhibition. We also present a novel method for characterization of the materials using liquid chromatography mass spectrometry to quantify the analytes of interest.

Quantification of fatty acid and mineral levels of selected seeds, nuts, and oils in Northern Ghana.

The purpose of this study was to identify locally available foods that can be utilized by Northern Ghanaians to improve child growth status. An assortment of seeds, nuts and oils were collected from a local market, packaged in plastic containers, and shipped to the US for all analyses. Fatty acids (FAs) were extracted and derivatized to FA methyl esters prior to quantification by GC/MS. ANOVA were conducted on FA concentrations and Tukey's post hoc test was used to compare FA content. Food grade oils, particularly palm oil and shea butter, contained higher saturated and monounsaturated FAs than seeds or nuts. Soybean, was significantly higher in the essential omega-3 FA alpha-linolenic acid (2.98 mg/g), whereas neri seed (68.4 mg/g) and fermented dawadawa (seed; 56.3 mg/g) had significantly higher amounts of total polyunsaturated FAs than all other foods. Iron levels in soybean (353 mg/kg), neri (282 mg/kg) and fermented dawadawa (165 mg/kg) were also the highest of all foods. Together, these foods may be useful for future intervention to curb stunting and iron-deficiency anemia.

Association of whole blood n-6 fatty acids with stunting in 2-to-6-year-old Northern Ghanaian children: A cross-sectional study.

In Northern Ghana, 33% of children are stunted due to economic disparities. Dietary fatty acids (FA) are critical for growth, but whether blood FA levels are adequate in Ghanaian children is unknown. The objective of this study was to determine the association between whole blood FAs and growth parameters in Northern Ghanaian children 2-6 years of age. A drop of blood was collected on an antioxidant treated card and analyzed for FA composition. Weight and height were measured and z-scores were calculated. Relationships between FAs and growth parameters were analyzed by Spearman correlations, linear regressions, and factor analysis. Of the 307 children who participated, 29.7% were stunted and 8% were essential FA deficient (triene/tetraene ratio>0.02). Essential FA did not differ between stunted and non-stunted children and was not associated with height-for-age z-score (HAZ) or weight-for-age z-score (WAZ). In hemoglobin adjusted regression models, both HAZ and WAZ were positively associated with arachidonic acid (p≤0.01), dihomo-gamma-linolenic acid (DGLA, p≤0.05), docosatetraenoic acid (p≤0.01) and the ratio of DGLA/linoleic acid (p≤0.01). These data add to the growing body of evidence indicating n-6 FAs are critical in childhood linear growth. Our findings provide new insights into the health status of an understudied Northern Ghanaian population.

The effect of fish oil supplementation on brain DHA and EPA content and fatty acid profile in mice.

Supplementation with omega-3 (n-3) fatty acids may improve cognitive performance and protect against cognitive decline. However, changes in brain phospholipid fatty acid composition after supplementation with n-3 fatty acids are poorly described. The purpose of this study was to feed increasing n-3 fatty acids and characterise the changes in brain phospholipid fatty acid composition and correlate the changes with red blood cells (RBCs) and plasma in mice. Increasing dietary docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) did not alter brain DHA. Brain EPA increased and total n-6 polyunsaturated fatty acids decreased across treatment groups, and correlated with fatty acid changes in the RBC (r > 0.7). Brain cis-monounsaturated fatty acids oleic and nervonic acid (p < .01) and saturated fatty acids arachidic, behenic, and lignoceric acid (p < .05) also increased. These brain fatty acid changes upon increasing n-3 intake should be further investigated to determine their effects on cognition and neurodegenerative disease.

Increasing dietary EPA and DHA influence estimated fatty acid desaturase activity in systemic organs which is reflected in the red blood cell in mice.

Delta-5 (D5D) and delta-6 (D6D) desaturase are key enzymes in fatty acid (FA) metabolism. Dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may alter tissue FA composition via D5D and D6D. The purpose was to determine the relationship between dietary EPA + DHA, estimated desaturase activities of various tissues and the reflection of desaturase activity in the red blood cell (RBC). Mice were fed diets with increasing percent of energy from EPA + DHA. Phospholipid FA composition of heart, muscle, spleen, lung, adipose tissues and RBC were analysed. D5D and D6D enzyme activity estimates (EAE) were calculated as the ratio of 20:4/20:3 and 20:3/18:2, respectively. D5D EAE decreased in all tissues, except muscle, with increasing dietary EPA + DHA. RBC D5D EAE positively correlated with D5D EAE in all tissues. RBC D6D EAE positively correlated with muscle and inversely correlated with adipose D6D EAE. Our findings suggest differential influence of dietary EPA + DHA upon tissue desaturase activities.

Obesity, adipokines, and C-peptide are associated with distinct plasma phospholipid profiles in adult males, an untargeted lipidomic approach.

Obesity is associated with dysregulated lipid metabolism and adipokine secretion. Our group has previously reported obesity and adipokines are associated with % total fatty acid (FA) differences in plasma phospholipids. The objective of our current study was to identify in which complex lipid species (i.e., phosphatidylcholine, sphingolipids, etc) these FA differences occur. Plasma lipidomic profiling (n = 126, >95% Caucasian, 48-65 years) was performed using chromatographic separation and high resolution tandem mass spectrometry. The responses used in the statistical analyses were body mass index (BMI), waist circumference (WC), serum adipokines, cytokines, and a glycemic marker. High-dimensional statistical analyses were performed, all models were adjusted for age and smoking, and p-values were adjusted for false discovery. In Bayesian models, the lipidomic profiles (over 1,700 lipids) accounted for >60% of the inter-individual variation of BMI, WC, and leptin in our population. Across statistical analyses, we report 51 individual plasma lipids were significantly associated with obesity. Obesity was inversely associated lysophospholipids and ether linked phosphatidylcholines. In addition, we identify several unreported lipids associated with obesity that are not present in lipid databases. Taken together, these results provide new insights into the underlying biology associated with obesity and reveal new potential pathways for therapeutic targeting.

Relationship between Body Mass Index, C-Peptide, and Delta-5-Desaturase Enzyme Activity Estimates in Adult Males.

Obesity, in particular abdominal obesity, alters the composition of plasma and tissue fatty acids (FAs), which contributes to inflammation and insulin resistance. FA metabolism is modulated by desaturases and may affect adipokine and insulin secretion. Therefore, we examined relationships between adipokines, a marker of insulin production, and plasma FA desaturase enzyme activity estimates (EAEs) in obesity. Plasma phospholipid (PPL) FAs were isolated from 126 males (ages 48 to 65 years), derivatized, and analyzed using gas chromatography. Delta-6 desaturase (D6D) and delta-5 desaturase (D5D) EAEs were calculated as the ratio of PPL 20:3/18:2 and 20:4/20:3, respectively. In body mass index (BMI) and waist circumference (WC) adjusted polytomous logistic regression analyses, PPL FAs and FA desaturase EAEs were associated with C-peptide and adiponectin. Individuals with elevated D6D EAEs were less likely (OR 0.33) to have serum adiponectin concentrations > 5.37 µg/mL, compared with adiponectin concentrations ≤ 3.62 µg/mL. Individuals with increased D5D EAEs were less likely (OR 0.8) to have C-peptide concentrations ≥ 3.32 ng/mL, and > 1.80 and ≤ 3.29 ng/mL, compared with those with C-peptide ≤ 1.76 ng/mL. The proinflammatory cytokine tumor necrosis factor-α (TNF-α) was positively associated with C-peptide, but TNF-α was not associated with the D5D EAE. C-peptide and adiponectin concentrations are associated with specific PPL FAs and FA desaturase EAEs. The relationship between C-peptide concentrations and D5D EAEs remained significant after adjusting for BMI, WC, and TNF-α. Thus, future research should investigate whether D5D inhibition may occur through a C-peptide mediated pathway.

Altered Saturated and Monounsaturated Plasma Phospholipid Fatty Acid Profiles in Adult Males with Colon Adenomas.

BACKGROUND: Altered lipid metabolism and plasma fatty acid (FA) levels are associated with colorectal cancer. Obesity and elevated waist circumference (WC) increase the likelihood of developing precancerous colon adenomas. METHODS: Venous blood was collected from 126 males, ages 48 to 65 years, who received routine colonoscopies. Plasma phospholipid (PPL) FAs were isolated, derivatized, and then analyzed using gas chromatography. ORs and 95% confidence intervals were determined using polytomous logistic regression after adjusting for confounding factors [i.e., age, smoking, WC, and body mass index (BMI)]. RESULTS: PPL palmitic acid (PA) was inversely correlated with the presence of colon adenomas (P = 0.01). For each unit increase in palmitoleic acid (OR, 3.75; P = 0.04) or elaidic acid (OR, 2.92; P = 0.04), an individual was more likely to have adenomas relative to no colon polyps. Higher enzyme activity estimates (EAE) of stearoyl-CoA desaturase-1 (SCD-1; P = 0.02) and elongation of very long chain fatty acids protein-6 (ELOVL-6; P = 0.03) were associated with an individual being approximately 1.5 times more likely to have an adenoma compared with no polyps. CONCLUSIONS: PPL FAs and EAEs, which have previously been associated with colorectal cancer, are significantly different in those with adenomas when compared with those without polyps. PPL PA, elaidic acid, and SCD-1 and ELOVL-6 EAEs are associated with adenomas independent of BMI and WC. IMPACT: PPL PA, elaidic acid, and SCD-1 and ELOVL-6 EAEs are associated with adenomas even after adjusting for obesity-related risk factors and may function as novel biomarkers of early colorectal cancer risk.

Plasma phospholipids, non-esterified plasma polyunsaturated fatty acids and oxylipids are associated with BMI.

The obese lipid profile is associated with increased free fatty acids and triacylglycerides. Currently, little is known about the plasma lipid species associated with obesity. In this study, we compared plasma lipid fatty acid (FA) profiles as a function of BMI. Profiling phospholipid (PL) FAs and their respective oxylipids could predict which obese individuals are more likely to suffer from diseases associated with chronic inflammation or oxidative stress. We investigated the relationship between BMI and plasma PL (PPL) FA composition in 126 men using a quantitative gas chromatography analysis. BMI was inversely associated with both PPL nervonic and linoleic acid (LA) but was positively associated with both dihomo-γ-linolenic and palmitoleic acid. Compared to lean individuals, obese participants were more likely to have ω-6 FAs, except arachidonic acid and LA, incorporated into PPLs. Obese participants were less likely to have EPA and DHA incorporated into PPLs compared to lean participants. Non-esterified plasma PUFA and oxylipid analysis showed ω-6 oxylipids were more abundant in the obese plasma pool. These ω-6 oxylipids are associated with increased angiogenesis (i.e. epoxyeicosatrienoates), reactive oxygen species (i.e. 9-hydroxyeicosatetraenoate), and inflammation resolution (i.e. Lipoxin A4). In summary, BMI is directly associated with specific PPL FA and increased ω-6 oxylipids.