A robust LC-MS/MS method to measure 8-oxoGuo, 8-oxodG, and NMN in human serum and urine.

OBJECTIVE: To establish and validate a robust LC-MS/MS method for simultaneously measuring 8-oxoGuo, 8-oxodG, and NMN in serum and urine to evaluate the oxidative stress status. METHODS: A Waters TQ-XS triple quadrupole mass spectrometer system coupled with an Acquity UPLC Primer HSS T3 column was chosen. The clinical performance was verified according to the CLSI C62-A and EP-15 guidelines. Furthermore, matched serum and urine samples from 22 apparently healthy check-ups, 20 patients with atherosclerosis, and 18 individuals with dementia were evaluated. RESULTS: The recovery for serum 8-oxoGuo, urine 8-oxoGuo, serum 8-oxodG, urine 8-oxodG, serum NMN, and urine NMN was 88.8-112.4%, 102.4-114.1%, 88.5-107.7%, 94.9-102.6%, 98.4-108.9%, and 88.5-108.6%, respectively. Based on the inter-assay results, total coefficient of variation, matrix effect, and carryover, the LC-MS/MS method was deemed robust. The limit of quantification was 0.017, 0.018, and 0.150 nmol/L for 8-oxoGuo, 8-oxodG, and NMN, respectively, which are suitable for accurate measurements in human serum and urine samples. Higher 8-oxoGuo and 8-oxodG levels and lower NMN levels, indicative of significantly higher oxidative stress status, were found in patients with dementia compared to healthy subjects. CONCLUSION: We established and validated a robust LC-MS/MS method to simultaneously measure 8-oxoGuo, 8-oxodG, and NMN in serum and urine.

Nightshift work can induce oxidative DNA damage: a pilot study.

BACKGROUND: Regular sleep is very important for human health; however, the short-term and long-term effects of nightshift with sleep deprivation and disturbance on human metabolism, such as oxidative stress, have not been effectively evaluated based on a realistic cohort. We conducted the first long-term follow-up cohort study to evaluate the effect of nightshift work on DNA damage. METHODS: We recruited 16 healthy volunteers (aged 33 ± 5 years) working night shifts at the Department of Laboratory Medicine at a local hospital. Their matched serum and urine samples were collected at four time points: before, during (twice), and after the nightshift period. The levels of 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), two important nucleic-acid damage markers, were accurately determined based on a robust self-established LC‒MS/MS method. The Mann-Whitney U or Kruskal-Wallis test was used for comparisons, and Pearson's or Spearman's correlation analysis was used to calculate the correlation coefficients. RESULTS: The levels of serum 8-oxodG, estimated glomerular filtration rate-corrected serum 8-oxodG, and the serum-to-urine 8-oxodG ratio significantly increased during the nightshift period. These levels were significantly higher than pre-nightshift work level even after 1 month of discontinuation, but no such significant change was found for 8-oxoG. Moreover, 8-oxoG and 8-oxodG levels were significantly positively associated with many routine biomarkers, such as total bilirubin and urea levels, and significantly negatively associated with serum lipids, such as total cholesterol levels. CONCLUSION: The results of our cohort study suggested that working night shifts may increase oxidative DNA damage even after a month of discontinuing nightshift work. Further studies with large-scale cohorts, different nightshift modes, and longer follow-up times are needed to clarify the short- and long-term effects of night shifts on DNA damage and find effective solutions to combat the negative effects.

An automated magnetic bead extraction method for measuring plasma metanephrines and 3-methoxytyramine using liquid chromatography tandem mass spectrometry.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is used routinely in clinical diagnostics; however, automating the sample pretreatment is challenging. We established and evaluated an automated method based on the magnetic bead extraction principle (MBE) to measure normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (3-MT). The target analytes were extracted, purified, and concentrated using different solvents and chemical bond-modified magnetic beads transferred via a magnetic bar. The linearity, recovery, matrix effect, and precision of MBE were evaluated thoroughly, and compared with traditional solid-phase extraction (SPE) using 131 plasma samples. The chromatography peaks of metanephrines and 3-MT, extracted via MBE, are symmetrical, without interfering peaks. The linearity was excellent with correlation coefficient (r) > 0.99. The MBE exhibited good reproducibility with within-run coefficient variations (CVs) of 1.96-2.00%, 4.06-5.75%, and 3.89-4.90% for MN, NMN, and 3-MT, respectively. The total CVs for MN, NMN, and 3-MT were 1.96-2.80%, 5.12-5.75%, and 5.44-6.27%, respectively. The relative recoveries for MN, NMN, and 3-MT varied between 93.5 and 107.4%, whereas their biases were all within 10%. The results for MN, NMN, and 3-MT extracted via MBE compared with SPE exhibited excellent correlation, with r > 0.99; the mean bias% for MN, NMN, and 3-MT were small (-2.9%, -3.2%, and -3.2%, respectively). In conclusion, the automated MBE method for measuring plasma metanephrines and 3-MT can be applied in future routine clinical diagnostics, and the MBE principle may indicate a new era for LC-MS/MS in clinical application.

Analytical and Clinical Performance of a Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Gastrin Subtypes G34 and G17 in Serum.

BACKGROUND: Two major forms of gastrin, gastrin-17 (G17) and gastrin-34 (G34), exist in blood. However, conventional immunoassay methods can only quantify total gastrin or G17 alone. Here, we aimed to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify G17 and G34 simultaneously. METHODS: Serum samples were prepared by anion-exchange solid-phase extraction. The analytical performance of the LC-MS/MS method was validated and the method was compared to chemiluminescence immunoassay (CLIA) and radioimmunoassay (RIA). The G17 and G34 concentrations in 245 serum samples from healthy controls, individuals with gastrinoma, and individuals with other diseases were analyzed. RESULTS: The total runtime of the LC-MS/MS method was 6 min. No substantial matrix effect was observed with internal standard correction. The intraassay coefficients of variation (CVs) for G17 and G34 were 4.0%-14.2% and 4.4%-10.4%, respectively, and total CVs were 5.2%-14.1% and 4.6%-12.4%, respectively. The correlation coefficient between LC-MS/MS and CLIA was 0.87, and between LC-MS/MS and RIA was 0.84. The G17+G34 concentrations for 87.5% of individuals with gastrinoma were higher than the 95th percentile of healthy controls (18.1 pg/mL), whereas the concentrations for individuals with other diseases and gastrinoma overlapped. Based on the Youden indices calculated for G17+G34, G34, and G17, the most specific biomarker was G17 (96.9% clinical specificity at 209.8 pg/mL) for gastrinoma. CONCLUSIONS: This method should aid in the diagnosis of diseases associated with increased gastrin concentrations.

Plasma or serum, which is the better choice for the measurement of metanephrines?

Measurement of metanephrines (MNs: metanephrine [MN] and normetanephrine [NMN]) is recommended for the initial biochemical diagnosis of pheochromocytoma and paraganglioma. Despite some drawbacks, plasma is commonly used for sampling. Here, we determined the feasibility of using serum, as an alternative to plasma, by comparing MNs in plasma and serum and evaluating the stability of MNs in serum. MNs obtained from serum, EDTA plasma, and heparin plasma were measured using LC-MS/MS immediately or after storage at 4 °C for 24 h, 72 h, and 7 days, and at -80 °C for 7 days, after sample collection. The differences between sample stability at given time points were compared using one-way ANOVA and Students' paired t-test, and the mean percent deviation was compared with total change limit (TCL). No significant difference was observed in MN and NMN between serum and EDTA plasma, and the mean percent deviation of the results obtained from serum compared to that from EDTA plasma was within the TCL. However, the difference of MN between EDTA plasma and heparin plasma exceeded the TCL. Both MNs in EDTA plasma and heparin plasma showed a significant decreasing trend at 4 °C with time (p < .01), while those in serum were relatively stable, with the mean percent deviation not exceeding the TCL at any time point or temperature. In conclusion, MNs measurement did not significantly differ between EDTA plasma and serum when measured immediately after collection, and MNs in serum were more stable than that in plasma.

Short-term biological variation and Reference change values of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine.

AIM: The DNA and RNA oxidative damage products urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-odGsn) and 8-oxo-7,8-dihydroguanosine (8-oGsn) have potential use in clinical practice. However, biological variation (BV) and reference change values (RCVs) have not been established. The aim of this study was to establish the short-term between-subject BV(CVG ), within-subject BV(CVI ), and RCVs for urinary 8-odGsn and 8-oGsn. METHODS: First-morning midstream urine specimens were collected from 20 apparently healthy subjects(ten males and ten females) on five consecutive days. 8-odGsn and 8-oGsn were measured using LC-MS/MS, while urine creatinine (U-Cr) was also measured to correct their results. A two-level nested ANOVA was used to estimate the CVI and CVG. RESULTS: The values of CVG for 8-odGsn, 8-odGsn/U-Cr, 8-oGsn, and 8-oGsn/U-Cr were 31.2%, 39.6%, 35.3%, and 28.8%, respectively, while CVI for them were 40.5%, 9.0%, 33.5%, and 12.1%, respectively. The RCVs for 8-odGsn, 8-odGsn/U-Cr, 8-oGsn, and 8-oGsn/U-Cr were 112.5%, 26.7%, 93.7%, and 36.5%, respectively. CONCLUSION: BV and RCVs were firstly established for 8-oxo-dGsn and 8-oGsn, and can be used in clinical practice.

Pre-analytical stability and physiological fluctuations affect plasma steroid hormone outcomes: A real-world study.

Since steroids are crucial for diagnosing endocrine disorders, the lack of research on factors that affect hormone levels makes interpreting the results difficult. Our study aims to assess the stability of the pre-analytical procedure and the impact of hormonal physiological fluctuations using real-world data. The datasets were created using 12,418 records from individuals whose steroid hormone measurements were taken in our laboratory between September 2019 and March 2024. 22 steroid hormones in plasma by a well-validated liquid chromatography tandem mass spectrometry method were measured. After normalization transformation, outlier removal, and z-score normalization, generalized additive models were constructed to evaluate preanalytic stability and age, sex, and sample time-dependent hormonal fluctuations. Most hormones exhibit significant variability with age, particularly steroid hormone precursors, sex hormones, and certain corticosteroids such as aldosterone. 18-hydroxycortisol, 18-oxocortisol. Sex hormones varied between males and females. Levels of certain hormones, including cortisol, cortisone, 11-deoxycortisol, 18-hydroxycortisol, 18-oxocortisol, corticosterone, aldosterone, estrone, testosterone, dihydrotestosterone, dehydroepiandrosterone sulfate, 11-ketotestosterone, and 11-hydroxytestosterone, fluctuated with sampling time. Moreover, levels of pregnenolone and progesterone decreased within 1 hour of sampling, with pregnenolone becoming unstable with storage time at 4 degrees after centrifugation, while other hormone levels remained relatively stable for a short period of time without or after centrifugation of the sample. This is the first instance real-world data has been used to assess the pre-analytic stability of plasma hormones and to evaluate the impact of physiological factors on steroid hormones.

A comprehensive LC-MS/MS method for the simultaneous measurement of 24 adrenal steroids: From research to clinical practice.

Steroids are essential in the differential diagnosis of congenital adrenal hyperplasia (CAH) subtypes; however, they may confuse physicians with multifarious results. In this study, we established a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of 24 steroids and developed a steroid metabolite pathway-based report to aid physicians in understanding these results. Solid-phase extraction was used to concentrate and purify target plasma steroids. The linearity, precision, recovery, and matrix effects were thoroughly evaluated. PowerBuilder was used to transfer the results from LC-MS/MS to the graphic report in a laboratory information management system (LIS) and was applied to different subtypes of CAH. Twenty-four steroids were separated and analyzed in one sample preparation and two injections using LC-MS/MS. The linearity of the steroids was excellent, with coefficients of linear regression greater than 0.99. The relative recovery ranged from 90.0 to 107.1 %, whereas the intra- and total coefficient variations were 1.6 âˆ¼ 8.7 % and 2.0 âˆ¼ 9.9 %, respectively. Matrix effects were compensated after internal standard correction. A graphic combination report mode was established and used to effectively identify CAH subtypes. In conclusion, a useful LC-MS/MS method and graphic combination report of 24 steroids based on their metabolite pathways were established.

Reference intervals for LC-MS /MS measurements of plasma renin activity, aldosterone, angiotensin II, and 24-hour urinary aldosterone in Northern Chinese Han population.

BACKGROUND: Examination of aldosterone to Renin Ratio (ARR) and plasma aldosterone concentration (PAC) or 24-h urinary aldosterone excretion (24-h UALD) was the necessary tests in confirmatory tests for primary aldosteronism (PA). We developed a combined liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for plasma renin activity (PRA), PAC, and angiotensin II (Ang II) and investigated their reference intervals (RIs) in northern Chinese Han population. The RIs of 24-h UALD excretion were also studied using LC-MS/MS. METHODS: A total of 309 healthy volunteers were recruited in 3 cities in China. PRA, PAC, Ang II, and 24-h UALD were measured using the laboratory-developed LC-MS/MS. Multiple linear regression and the variance component model were applied to determine if the RI needed to be split. The RIs of PRA, PAC, and Ang II were determined using the nonparametric percentile method. RESULTS: The laboratory-developed LC-MS/MS method was verified and showed good performance. Standard deviation ratio (SDR) sex for PAC and SDR region for Ang II are 0.466 and 0.407, respectively, indicating that the RIs of PAC and Ang II must be divided by sex and region, respectively. In addition, the SDR 24hUK for 24-h UALD is 0.579, indicating that the RI of 24-h UALD must be partitioned by urine potassium. CONCLUSION: RIs were established for tests related to the renin-angiotensin-aldosterone system in the apparently healthy northern Chinese Han population by the LC-MS/MS method.

A robust method for simultaneous measurement of serum 25(OH)D, 1,25(OH)2 D, and 24,25(OH)2 D by liquid chromatography-tandem mass spectrometry with efficient separation of 3-epi analogs, 23R,25(OH)2 D3 , and 4ß,25(OH)2 D3.

BACKGROUND: This study aimed to establish a robust, simple method to detect 25-hydroxyvitamin D3 (25(OH)D3 ), 25-hydroxyvitamin D2 (25(OH)D2 ), 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), 1,25-dihydroxyvitamin D2 (1,25(OH)2 D2 ), 24,25-dihydroxyvitamin D3 (24,25(OH)2 D3 ), and 24,25-dihydroxyvitamin D2 (24,25(OH)2 D2 ) simultaneously with efficient separation of 3-epi 25(OH)D3 , 3-epi 24,25(OH)2 D3 , 23R,25(OH)2 D3 , and 4ß,25-dihydroxyvitamin D3 (4ß,25(OH)2 D3 ) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHOD: This method was validated according to procedures established by Clinical and Laboratory Standards Institute (CLSI) and then applied in healthy population to determine the distribution of the vitamin D metabolites by LC-MS/MS. RESULTS: The total-run CV% of 25(OH)D3 , 25(OH)D2 , 24,25(OH)2 D3 , 24,25(OH)2 D2 , 1,25(OH)2 D3 , and 1,25(OH)2 D2 were 6.30%-8.40%, 5.00%-8.40%, 5.90%-9.00%, 5.60%-9.00%, 5.60%-8.00%, and 7.00%-9.70%, respectively. The linearity correlation coefficients r of these six vitamin D metabolites were >0.99. The matrix effects of 25(OH)D3 , 25(OH)D2 , 24,25(OH)2 D3 , 24,25(OH)2 D2 , 1,25(OH)2 D3 , and 1,25(OH)2 D2 were 90.6%-103.3%, 97.3%-106.3%, 90.7%-106.3%, 100.7%-114.5%, 97.9%-104.6%, and 97.0%-111.0%. The trueness values of 25(OH)D3 , 25(OH)D2 , and 24,25(OH)2 D3 were 93.8%-103.0%, 101.0%, and 96.3%-100%, respectively. CONCLUSION: This study successfully established an efficient, accurate, robust method for simultaneous measurement of serum 25(OH)D, 1,25(OH)2 D, and 24,25(OH)2 D by LC-MS/MS with efficient separation of 3-epi analogs, 23R,25(OH)2 D3 , and 4ß,25(OH)2 D3 .

Endocrine pheromones couple fat rationing to dauer diapause through HNF4α nuclear receptors.

Developmental diapause is a widespread strategy for animals to survive seasonal starvation and environmental harshness. Diapaused animals often ration body fat to generate a basal level of energy for enduring survival. How diapause and fat rationing are coupled, however, is poorly understood. The nematode Caenorhabditis elegans excretes pheromones to the environment to induce a diapause form called dauer larva. Through saturated forward genetic screens and CRISPR knockout, we found that dauer pheromones feed back to repress the transcription of ACOX-3, MAOC-1, DHS-28, DAF-22 (peroxisomal ß-oxidation enzymes dually involved in pheromone synthesis and fat burning), ALH-4 (aldehyde dehydrogenase for pheromone synthesis), PRX-10 and PRX-11 (peroxisome assembly and proliferation factors). Dysfunction of these pheromone enzymes and factors relieves the repression. Surprisingly, transcription is repressed not by pheromones excreted but by pheromones endogenous to each animal. The endogenous pheromones regulate the nuclear translocation of HNF4α family nuclear receptor NHR-79 and its co-receptor NHR-49, and, repress transcription through the two receptors. The feedback repression maintains pheromone homeostasis, increases fat storage, decreases fat burning, and prolongs dauer lifespan. Thus, the exocrine dauer pheromones possess an unexpected endocrine function to mediate a peroxisome-nucleus crosstalk, coupling dauer diapause to fat rationing.

Sources of variation evaluation of 24,25(OH)2D levels and the ratio of 25OHD to 24,25(OH)2D in apparently healthy Chinese adults: a multicenter cross-sectional study.

24,25(OH)2D and the 25OHD/24,25(OH)2D ratio have recently been shown to be useful for screening for hypercalcemia caused by CYP24A1 mutations. However, no population-based data on 24,25(OH)2D have been published for Chinese populations, and few studies evaluated the sources of variation on 24,25(OH)2D levels and the 25OHD/24,25(OH)2D ratio. Hence, in 2018, we enrolled 1211 apparently healthy adults without systematic diseases from six representative cities in China (Beijing, Dongying, Guiyang, Urumqi, Shenzhen, and Qiqihar). 24,25(OH)2D and 25OHD levels were measured using isotope dilution liquid chromatography tandem mass spectrometry. Multiple regression analysis showed that sex contributed the most to variations in 24,25(OH)2D, 25OHD, and 25OHD/24,25(OH)2D (rp= -0.255, -0.253, and 0.141, respectively), and age also contributed to variations in 25OHD but not 24,25(OH)2D or 25OHD/24,25(OH)2D. Men had significantly higher 24,25(OH)2D and 25OHD levels than women, but had significantly lower 25OHD/24,25(OH)2D values. Individuals from Dongying had the highest 24,25(OH)2D and 25OHD values, whereas individuals from Urumqi had the lowest values. The median(2.5-97.5%) values for 24,25(OH)2D, 25OHD, and 25OHD/24,25(OH)2D were 1.2(0.36-2.65) ng/mL, 18.9(8.6-32.5) ng/mL, and 16.0 (9.8-30.8), respectively. 24,25(OH)2D was significantly correlated with 25OHD (r = 0.838, p <  0.001), and 25OHD/24,25(OH)2D was significantly negatively correlated with 24,25(OH)2D (r = -0.758, P < 0.001) and 25OHD (r = -0.310, P < 0.001). In conclusion, in this nationwide, multicenter, cross-sectional study, we evaluated the levels of 24,25(OH)2D and the 25OHD/24,25(OH)2D ratio in the Chinese population. Sex contributed the most to variations in 24,25(OH)2D, 25OHD, and 25OHD/24,25(OH)2D.

Rapid liquid chromatography-tandem mass spectrometry to determine very-long-chain fatty acids in human and to establish reference intervals for the Chinese population.

Very-long-chain fatty acids (VLCFAs), including hexacosanoic, tetracosanoic, and docosanoic acids, are peroxisomal disease markers, whose abnormal accumulation warrants prompt detection for timely, effective treatment. This study aimed to establish and validate a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method to simultaneously quantify VLCFAs and provide reference intervals among Chinese individuals, quantify VLCFAs in pregnancy, and explore potential associations between plasma and amniotic fluid. Analytes were extracted via water-bath incubation with HCl and liquid-liquid extraction. Method linearity, limit of detection/quantitation, precision, carryover, and recovery were evaluated according to Clinical and Laboratory Standard Institute (CLSI) guidelines. VLCFAs showed good reproducibility based on low within-run coefficient variations (CVs) and total CVs, and correlation coefficients of linearity were > 0.99. The reference interval of C22:0, C24:0, and C26:0 were 32.0-73.4 µmol/L, 30.3-72.0 µmol/L, and 0.20-0.71 µmol/L, respectively; C24:0/C22:0 and C26:0/C22:0 ratios were 0.75-1.28 and 0.005-0.0139, respectively. Plasma and amniotic fluid of the same pregnant women displayed no significant correlation in the second trimester. This study presents the simple, efficient, accurate, and robust LC-MS/MS method to simultaneously detect C22:0, C24:0, and C26:0 without derivatization; it can be used to establish reference intervals among Chinese individuals and has diagnostic and other clinical applications.

Validation of an improved liquid chromatography tandem mass spectrometry method for rapid and simultaneous analysis of plasma catecholamine and their metabolites.

Catecholamines [dopamine (DA), epinephrine (E), and norepinephrine (NE)] and their metabolites [metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT)] are functionally important in humans. Their overexpression can indicate the presence of neuroendocrine tumors. Accurate and rapid quantitation of catecholamines and their metabolites may function in differential diagnosis of neuroendocrine tumors. Herein, we diluted 200 µL plasma using isotope labelled internal standards (IS), and extracted using solid phase extraction. The performance of isotope diluted liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) was evaluated and applied to quantify the level of catecholamines and metabolites in clinical samples from 73 apparently healthy adults. The total analysis time of the ID-LC-MS/MS method was 4 min. The improved method was highly sensitive, with a limit of quantification (LOQ) for MN, NMN, 3-MT, and E of 1 pg/mL, a LOQ for DA of 5 pg/mL, and for NE of 10 pg/mL. After correction using IS, no significant matrix effects were observed. Good reproducibility was obtained, with total CVs of 3.2-13.1% (DA), 4.8-10.0% (E), 6.2-6.9% (NE), 3.8-7.9% (MN), 4.1-8.8% (NMN), 3.4-8.9% (3-MT). Recoveries were in the range of 91.1-109.7% for the six analytes. Also, the mean concentration of catecholamines were as follows: MN, 22.9 ±â€¯7.2 pg/mL; NMN, 41.4 ±â€¯17.2 pg/mL; 3-MT, 2.34 ±â€¯2.01 pg/mL; DA, 10.2 ±â€¯4.6 pg/mL; E, 29.3 ±â€¯14.2 pg/mL and NE 427.0 ±â€¯190.6 pg/mL. A reliable ID-LC-MS/MS method for the determination of catecholamines and their metabolites using small volumes of plasma was verified. This method is rapid, simple, and may serve as an essential diagnostic tool for neuroendocrine tumors in clinical practice.

A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans.

To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 µm in diameter. From a saturated forward genetic screen of 6.7 × 10(5) mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 µm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion.