Quantitation of urinary methylmalonic acid by gas chromatography mass spectrometry and its clinical applications

Urine methylmalonic acid (MMA) concentrations were detected in 79 Chinese patients by gas chromatography mass spectrometry (GC/MS), using a selected ion monitoring program. 10 of the 79 patients were found to have cobalamin deficiency. Their urine MMA amounts were all elevated with a mean value 1376 ng/microliter (11.66 mmol/l), ranging from 40.46 to 3900 ng/microliter (0.34-33.05 mmol/l). The remaining 69 cases were found to be unrelated to cobalamin deficiency. Their mean urine MMA was 3.62 ng/microliter (30.0 mumol/l), ranging from 0-17.47 ng/microliter (0-148.0 mumol/l). In this study, we found that urine MMA detected by GC/MS was a simple, rapid, convenient, specific and sensitive method for the diagnosis of cobalamin deficiency. The urine MMA concentrations in cases not due to cobalamin deficiency would not exceed 20 ng/microliter (169.5 mumol/l), whereas in cobalamin deficiency the urine MMA levels always exceeded 20 ng/microliter, or were even much higher. No overlapping of the results of urine MMA between these 2 groups of patients could be seen in our study. Detection of urine MMA is useful in the demonstration or exclusion of cobalamin deficiency in any suspect patients.     

Detection of inborn errors of metabolism in Thai infants via gas chromatography and mass spectrometry

We had studied inherited metabolic disorders at the Department of Pediatrics, Siriraj Hospital Faculty of Medicine, Mahidol University since 1987 using limited resources available and collaboration with other laboratories, both in Thailand and The United States. Since April 1998, we started a collaboration with MILS and Kanazawa Medical University, the Japan, studying inborn errors of metabolism in Asian Countries using urine filter paper and a new GC/MS method. We have since successfully discovered several patients with metabolic disorders. Out of 33 (high-risk) cases we sent for biochemical diagnosis (during April-July 1998), 13 abnormal results were found which is approximately 39.4%. Inherited metabolic disorders identified were as follows: medium-chain acyl CoA dehydrogenase deficiency (MCAD), multiple carboxylase deficiency (MCD), methylmalonic acidemia (MMA), Fanconi syndrome, galactosemia and neuroblastoma.     

The diagnosis of congenital adrenal hyperplasia in the newborn by gas chromatography/mass spectrometry analysis of random urine specimens

Definitive neonatal diagnosis of congenital adrenal hyperplasia (CAH) is frequently complicated by normal 17-hydroxyprogesterone levels in 21-hydroxylase-deficient patients, residual maternal steroids, and other interfering substances in neonatal blood. In an effort to improve the diagnosis, we developed a gas chromatography/mass spectrometry method for simultaneous measurement of 15 urinary steroid metabolites as early as the first day of life. Furthermore, we developed 11 precursor/product ratios that diagnose and clearly differentiate the four enzymatic deficiencies that cause CAH. Random urine samples from 31 neonatal 21-hydroxylase-deficient patients and 59 age-matched normal newborns were used in the development. Additionally, samples from two 11 beta-hydroxylase-deficient patients and one patient each for 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase deficiencies were used. The throughput for one bench-top gas chromatography/mass spectrometry instrument is 20 samples per day. Thus, this method affords an accurate, rapid, noninvasive means for the differential diagnosis of CAH in the newborn period without the need for invasive testing and ACTH stimulation.     

Diagnosis of pulmonary tuberculosis by detection of tuberculostearic acid in sputum by using gas chromatography-mass spectrometry with selected ion monitoring

Tuberculostearic acid [(R)-10-methyloctadecanoic acid (TBSA)] is a structural component of mycobacteria, and its detection in appropriate clinical specimens has potential application as a rapid screening method for the presence of Mycobacterium tuberculosis and other mycobacteria. We used the highly sensitive technique of gas chromatography-mass spectrometry (GC-MS) combined with selected ion monitoring (SIM) of m/e 312 and m/e 167 for detecting TBSA in 405 clinical sputum specimens collected in Hong Kong, where tuberculosis is still common. TBSA was detected in 39 M. tuberculosis smear-positive, culture-positive specimens; 63 of 66 smear-negative, culture-positive specimens; and 1 of 300 smear-negative, culture-negative specimens. Thus, for screening of sputa from individuals with suspected pulmonary tuberculosis, the detection of TBSA by GC-MS/SIM is highly specific and more sensitive than conventional microscopy and more rapid but slightly less sensitive than conventional culture methods.     

Measurement of breath acetone concentrations by selected ion flow tube mass spectrometry in type 2 diabetes

Selected ion flow tube-mass spectrometry (SIFT-MS) can measure volatile compounds in breath on-line in real time and has the potential to provide accurate breath tests for a number of inflammatory, infectious and metabolic diseases, including diabetes. Breath concentrations of acetone in type 2 diabetic subjects undertaking a long-term dietary modification programme were studied. Acetone concentrations in the breath of 38 subjects with type 2 diabetes were determined by SIFT-MS. Anthropomorphic measurements, dietary intake and medication use were recorded. Blood was analysed for beta hydroxybutyrate (a ketone body), HbA1c (glycated haemoglobin) and glucose using point-of-care capillary (fingerprick) testing. All subjects were able to undertake breath manoeuvres suitable for analysis. Breath acetone varied between 160 and 862 ppb (median 337 ppb) and was significantly higher in men (median 480 ppb versus 296 ppb, p = 0.01). In this cross-sectional study, no association was observed between breath acetone and either dietary macronutrients or point-of-care capillary blood tests. Breath analysis by SIFT-MS offers a rapid, reproducible and easily performed measurement of acetone concentration in ambulatory patients with type 2 diabetes. The high inter-individual variability in breath acetone concentration may limit its usefulness in cross-sectional studies. Breath acetone may nevertheless be useful for monitoring metabolic changes in longitudinal metabolic studies, in a variety of clinical and research settings.     

Injury elicited increase in spinal cord neurosteroid content analyzed by gas chromatography mass spectrometry

The effects of spinal cord injury (SCI), combined with castration and adrenalectomy, and of progesterone (PROG) treatment on neurosteroid levels and steroidogenic enzyme expression were investigated in the adult male rat spinal cord (SC). Steroid levels were quantified by gas chromatography/mass spectrometry in SC and plasma, and mRNAs of enzymes by quantitative real-time RT-PCR. The levels of pregnenolone (PREG), PROG, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone increased in SC 75 h after transection without significant increase in the plasma. After combined adrenalectomy and gonadectomy, significant levels of PREG and PROG remained in the SC, suggesting their local biosynthesis. In the SC of adrenalectomized and gonadectomized rats, there was an increase of PREG 24 h after SCI, followed at 75 h by a concomitant increase in its direct metabolite, PROG. These observations are consistent with a sequential increase of PREG biosynthesis and its conversion to PROG within the SC in response to injury. However, no significant change in P450-side chain cleavage and 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase mRNA levels was observed after SCI. Systemic PROG treatment after SCI, resulted in a very large increase in PROG, 5alpha-dihydroprogesterone, and 3alpha,5alpha-tetrahydroprogesterone in both plasma and SC. Furthermore, high levels of 3beta,5alpha-tetrahydroprogesterone were detected in SC, whereas their plasma levels remained barely detectable. Because the ratio of reduced metabolites to PROG was 65-times higher in SC than in the plasma, it appears likely that reduced metabolites mainly originated from local biosynthesis. Our results strongly suggest an important role for locally biosynthesized neurosteroids in the response of the SC to injury.     

Plasma galactose and galactitol concentration in patients with galactose-1-phosphate uridyltransferase deficiency galactosemia: determination by gas chromatography/mass spectrometry

The plasma concentration of galactose and galactitol was measured in 27 patients with galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia on a lactose-restricted diet, 17 infants on lactose-free formula, and 21 infants and children on a normal diet, by a newly devised isotope dilution gas chromatograph/mass spectrometry (GC/MS) method. The method was linear in the range of 0.1 to 10 micromol/L for galactose and 1 to 20 micromol/L for galactitol with good reproducibility and a coefficient of variation less than 3%. The mean plasma galactose in 15 patients who were homozygous for the most common Q188R mutation of the GALT gene was 2.72 +/- 0.70 micromol/L (mean +/- SE) with a range of 0.58 to 3.98 in specimens obtained at regular clinic visits. In 12 patients with other GALT mutations, it was 2.45 +/- 0.75 micromol/L. The mean value in nongalactosemic subjects on lactose-free formula was 0.52 +/- 0.08 micromol/L, with a range of 0.12 to 1.25. The range in 21 normal subjects without diet restriction was 0.11 to 6.33 micromol/L, with a mean of 1.48 +/- 0.32. The plasma galactitol level was 11.63 +/- 0.46 and 10.85 +/- 1.38 micromol/L in the 2 galactosemic groups. There was no relationship between plasma galactose and galactitol levels, with variable ratios of the two substances in the galactosemic patients. Galactitol was not detectable in the plasma of normal subjects. The red blood cell galactose-1-phosphate level was also measured in the galactosemic patients, and no relationship between plasma galactose and red blood cell galactose-1-phosphate was found. The galactose-1-phosphate concentration was 28 to 54 times higher than the ambient galactose. The low galactose concentration in the plasma of galactosemics on galactose-restricted diets in relation to the higher plasma galactitol and red blood cell galactose-1-phosphate is a metabolic enigma. The ability to measure plasma galactose accurately presents a new way of characterizing the galactosemic patient and the levels monitored over time may provide insight into the development of long-term complications associated with the disorder.     

A simple method for the estimation of a urinary steroids profile using glass capillary gas chromatography with a solventless injection system

A simple method for the estimation of a urinary steroids profile with glass capillary gas chromatography is described. A 10ml portion of a urinary specimen was hydrolysed by Helix pomatia juice. Additional hydrolysis with ox-liver beta glucuronidase was necessary for the complete liberation of the urinary steroids from their glucuronides conjugates. Liberated free steroids were extracted with ethyl acetate, and the extract was washed and evaporated under reduced pressure. Finally extracted steroids were derivatised as MO-TMS (methoxime-trimethylsilyl) ethers. For the full-silylation of the steroids, the Sakauchi-Hornig method was employed and good results were obtained. An OV-101-WCOT column (30m length) was connected to the "solventless injection system" (a type of glass falling needle injection) and temperature-programmed gas chromatography was performed. By this method, eight fractions of 17-KS, Pregnanediol, Pregnanetriol, Pregnanetriolone, 5-Pregnentriol, tetrahydro-metabolites of 11-Deoxy-Cortisol and of Cortisol, and hexahydrometabolites of Cortisol were separated and quantitatively determined. Data processing was performed by an on-line microcomputer. The value in normal male and female controls was compatible with the results of glass capillary gas chromatography measured by Shackleton. Urine specimens from children with adrenocortical cancer and 21-hydroxylase deficiency were analyzed. Characteristic metabolic profiles of each patient were easily demonstrated. This method seems suitable for the routine clinical elucidation of abnormal steroid metabolism.     

Steroid profiling in brain and plasma of male and pseudopregnant female rats after traumatic brain injury: analysis by gas chromatography/mass spectrometry

Steroids in brain arise from the peripheral endocrine glands and local synthesis. In traumatic brain injury (TBI), the endogenous circulating hormones at the time of injury are important for neuroprotection. In particular, pseudopregnant females recover better than males from TBI. We investigated the effect of pseudopregnancy and TBI on steroid levels in plasma and in three brain regions (within, adjacent, and distal to the lesion site), 6 and 24 h after prefrontal cortex injury. The following steroids were analyzed by gas chromatography/mass spectrometry: pregnenolone, progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, 3beta,5alpha-tetrahydroprogesterone, dehydroepiandrosterone, Delta(4)-androstenedione, testosterone, 5alpha-dihydrotestosterone, 3alpha,5alpha-tetrahydrotestosterone, 3beta,5alpha-tetrahydrotestosterone, and 17beta-estradiol. Corticosterone was assayed in plasma to account for stress in the rats. We found different steroid profiles in brain and plasma of male and pseudopregnant female rats and specific profile changes after TBI. In sham-operated pseudopregnant females, much higher levels of progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, and 3beta,5alpha-tetrahydroprogesterone were measured in both brain and plasma, compared with sham-operated males. Plasma levels of corticosterone were high in all groups, indicating that the surgeries induced acute stress. Six hours after TBI, the levels of pregnenolone, progesterone, and 5alpha-dihydroprogesterone increased, and those of testosterone decreased in male brain, whereas levels of 5alpha-dihydroprogesterone and 3beta,5alpha-tetrahydroprogesterone increased in brain of pseudopregnant female rats. Plasma levels of 5alpha-dihydroprogesterone did not change after TBI, suggesting a local activation of the 5alpha-reduction pathway of progesterone in both male and pseudopregnant female brain. The significant increase in neurosteroid levels in the male brain after TBI is consistent with their role in neuroprotection. In pseudopregnant females, high levels of circulating progestagens may provide protection against TBI.     

Reverse phase high-performance liquid chromatography and secondary ion mass spectrometry. A strategy for identification of ten human hemoglobin variants

Ten abnormal hemoglobins were detected and characterized in individual cases referred to our laboratory for evaluation of hematological problems. Six of these variants were electrophoretically silent and could be detected by reverse phase high-performance liquid chromatography (HPLC) analysis. HPLC was also used to analyze the tryptic peptides of each individual variant. In most of these variants, secondary ion mass spectra of the mixture of the tryptic peptides could reveal the aberrant peptide and predict possible substitution through the mass difference between the normal and abnormal peptide. The mass spectra of the isolated abnormal peptide generally contained sufficient fragment ions to define the position of the amino acid substitution, obviating the need for lengthy sequencing procedures. Combination of the two techniques.