Download mutations, and several investigators have characterized eight

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Technical Briefs
mutations, and several investigators have characterized
eight private or rare mutations (24 –26 ). Interestingly, a
recent report describes a full HFE screening procedure
using DHPLC methodology (27 ). Building on the same
technology, these two approaches could be complementary in a full evaluation of the HFE involvement in HC.
This would allow identification of iron-overloaded patients in whom HFE is not involved in the disease and
who are therefore candidates for the exploration of other
genes involved in iron-metabolism disorders.
22.
23.
24.
25.
26.
References
1. Worwood M. Genetics of haemochromatosis. Baillieres Clin Haematol
1994;7:903–18.
2. Niederau C, Fischer R, Purschel A, Stremmel W, Haussinger D, Strohmeyer
G. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996;110:1107–19.
3. Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A
novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399 – 408.
4. Feder JN, Penny DM, Irrinki A, Lee VK, Lebron JA, Watson N. The hemochromatosis gene product complexes with the transferrin receptor and lowers its
affinity for ligand binding. Proc Natl Acad Sci U S A 1998;95:1472–7.
5. Mura C, Raguenes O, Ferec C. HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis. Blood 1999;93:2502–5.
6. Bernard PS, Ajioka RS, Kushner JP, Wittwer CT. Homogeneous multiplex
genotyping of hemochromatosis mutations with fluorescent hybridization
probes. Am J Pathol 1998;1055– 61.
7. Bollhalder M, Mura C, Landt O, Maly FE. LightCycler PCR assay for
simultaneous detection of the H63D and S65C mutations in the HFE
hemochromatosis gene based on opposite melting temperature shifts. Clin
Chem 1999;45:2275– 8.
8. Walburger DK, Afonina IA, Wydro R. An improved real time PCR method for
simultaneous detection of C282Y and H63D mutations in the HFE gene
associated with hereditary hemochromatosis. Mutat Res 2001;432:69 –78.
9. Steinberg KK, Cogswell ME, Chang JC, Caudill SP, McQuillan GM, Bowman
BA, et al. Prevalence of C282Y and H63D mutations in the hemochromatosis (HFE) gene in the United States. JAMA 2001;285:2216 –22.
10. Donohoe GG, Laaksonen M, Pulkki K, Rönnemaa T, Kairisto V. Rapid
single-tube screening of the C282Y hemochromatosis mutation by real-time
multiplex allele-specific PCR without fluorescent probes. Clin Chem 2000;
46:1540 –7.
11. Liang Q, Davis PA, Thompson BH, Simpson JT. High-performance liquid
chromatography multiplex detection of two single nucleotide mutations
associated with hereditary hemochromatosis. J Chromatogr B 2001;754:
265–70.
12. Baty D, Kwiatkowski AT, Mechan D, Harris A, Pippard MJ, Goudie D.
Development of a multiplex ARMS test for mutations in the HFE gene
associated with hereditary haemochromatosis. J Clin Pathol 1998;51:
73– 4.
13. Mullighan CG, Bunce M, Fanning GC, Marshall SE, Welsh KI. A rapid method
of haplotyping HFE mutations and linkage disequilibrium in a Caucasoid
population. Gut 1998;42:566 –9.
14. Medintz I, Wong WW, Berti L, Shiow L, Tom J, Scherer J, et al. Highperformance multiplex SNP analysis of three hemochromatosis-related
mutations with capillary array electrophoresis microplates. Genome 2001;
11:413–21.
15. Lubin IM, Yamada NA, Stansel RM, Pace RG, Rohlfs EM, Silverman LM. HFE
genotyping using multiplex allele-specific polymerase chain reaction and
capillary electrophoresis. Arch Pathol Lab Med 1999;123:1177– 81.
16. Klingler KR, Zech D, Wielckens K. Haemochromatosis: automated detection
of HFE two point mutations in the HFE gene: Cys282Tyr and His63Asp. Clin
Chem Lab Med 2000;38:1225–30.
17. Beutler E, Gelbart T. Large-scale screening for HFE mutations: methodology
and cost. Genet Test 2000;4:131–7.
18. Oberkanins C, Moritz A, Villiers JNPD, Kotze MJ, Kury F. A reversehybridization assay for the rapid and simultaneous detection of nine HFE
gene mutations. Genet Test 2000;4:121– 4.
19. Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: a
review. Hum Mutat 2001;17:439 –74.
20. Kaler SG, Devaney JM, Pettit EL, Kirshman R, Marino MA. Novel method for
molecular detection of the two common hereditary hemochromatosis mutations. Genet Test 2000;4:125–9.
21. Gerry NP, Witowski NE, Day J, Hammer RP, Barany G, Barany F. Universal
27.
DNA microarray method for multiplex detection of low abundance point
mutations. J Mol Biol 1999;292:251– 62.
Rychlik W. Oligo6®, Ver. 6.57 for Windows. http://www.oligo.net (Accessed
August 2001).
Hansen NF, Oefner PJ. DHPLC Melt program. http://insertion.stanford.edu/
melt.html (Accessed August 2001).
Piperno A, Arosio C, Fossati L, Vigano M, Trombini P, Vergani A, et al. Two
novel nonsense mutations of HFE gene in five unrelated Italian patients with
hemochromatosis. Gastroenterology 2000;119:441–5.
Pointon JJ, Wallace D, Merryweather-Clarke AT, Robson KJH. Uncommon
mutations and polymorphisms in the hemochromatosis gene. Genet Test
2000;4:151– 61.
de Villiers JN, Hillermann R, Loubser L, Kotze MJ. Spectrum of mutations in
the HFE gene implicated in haemochromatosis and porphyria. Hum Mol
Genet 1999;8:1517–22.
Le Gac G, Mura C, Ferec C. Complete scanning of the hereditary hemochromatosis gene (HFE) by use of denaturing HPLC. Clin Chem
2001;47:1633– 40.
New Nuclear Encoded Mitochondrial Mutation Illustrates Pitfalls in Prenatal Diagnosis by Biochemical
Methods, Markus Schuelke,1* Anne Detjen,1 Lambert van den
Heuvel,2 Christoph Korenke,3 Antoon Janssen,2 Arie Smits,4
Frans Trijbels,2 and Jan Smeitink2 (1 Department of Neuropediatrics, Charité Virchow University Hospital, Augustenburger Platz 1, D-13353 Berlin, Germany; 2 Department of Pediatrics, Nijmegen Center for Mitochondrial
Disorders, University Medical Center Nijmegen, PO Box
9101, NL-6500 HB Nijmegen, The Netherlands; 3 Department of Neuropediatrics, Children’s Hospital, GeorgAugust-Universität, Robert-Koch Strasse 40, D-37075 Göttingen, Germany; 4 Department of Human Genetics,
University Medical Center, PO Box 9101, NL-6500 HB
Nijmegen, The Netherlands; * author for correspondence:
fax 49-30-4505-66920, e-mail [email protected])
A frequent etiology of congenital lactic acidosis is disturbed mitochondrial energy metabolism. Affected children generally present with neurologic symptoms, such
as myopathy and epilepsy. Parents who have lost a child
to mitochondrial disease often ask for prenatal diagnosis
in subsequent pregnancies. The large number of possible
mitochondrial or nuclear DNA mutations often makes the
molecular defect unknown. In these cases, prenatal diagnosis rests solely on biochemical analysis. Here we report
a possible pitfall in prenatal diagnosis of mitochondriopathies by biochemical methods that might occur despite all
precautions. It is illustrated by a patient with isolated
mitochondrial complex I deficiency and her family in the
light of a new mutation (632C3 T) in 1 of the 36 nuclear
encoded genes of complex I (NDUFV1).
The girl (II.1 in Fig. 1A) was the first child of healthy
Caucasian first-degree cousins. Postnatally she showed
acrocyanosis, muscular hypotonia, and a pendular nystagmus. Fundoscopy revealed bitemporal retinal depigmentation. The latencies of the visual evoked potentials
were pathologically increased. Lactic acidosis (pH 7.19)
was noted, with a plasma lactate concentration of 24.1
mmol/L (reference interval, 0.5–2.2 mmol/L), a lactate-
Clinical Chemistry 48, No. 5, 2002
to-pyruvate ratio of 57 (reference values ⬍20), plasma
alanine of 893 ␮mol/L (reference interval, 40 –500 ␮mol/
L), urine ␣-ketoglutaric acid of 1852 mmol/mol creatinine
(reference interval, 159 ⫾ 137 mmol/mol creatinine),
urine lactate of 1713 mmol/mol creatinine (reference
interval, 234 ⫾ 165 mmol/mol creatinine), and cerebrospinal fluid lactate of 9.6 mmol/L (reference values ⬍2
mmol/L). Cranial ultrasound and magnetic resonance
imaging results were normal. Muscle histology revealed
intracytoplasmic accumulation of glycogen. Mitochondria
were ultrastructurally normal on electron microscopy.
Fig. 1. Genetic analysis of the affected family.
(A), pedigree of the consanguineous family. (B), restriction endonuclease analysis of PCR products. When the 632C3 T mutation is present, a Bsp1268I site is
generated. Lane 1, DNA from cultured fibroblasts; lane 2, DNA from muscle
tissue from the index patient; lane 3, DNA from peripheral blood lymphocytes of
the mother; lane 4, DNA from cultured chorionic villi of the second pregnancy;
lane 5, DNA from chorionic villi of the aborted fetus; lane 6, DNA from peripheral
blood lymphocytes of the father; lane 7, DNA from uncultured chorionic villi of the
third pregnancy; lane 8, DNA from cultured chorionic villi of the third pregnancy;
lane 9, DNA from a healthy control. A 100-bp DNA reference ladder is shown on
both sides of the electropherogram. WT, wild type; MUT, mutant. (C), length
polymorphisms of 10 different markers, indicated on the right. The connecting
lines at the bottom depict identical haplotypes. The discrepancy between lanes
4 and 5 can be seen clearly. (D), alignment of NADH dehydrogenases of different
species. The A211V mutation occurs within a sequence motif that is highly
conserved between prokaryotic and eukaryotic species (GenBank accession nos.
AF053070, M58607, Z50109, X83999, M64432, and AE000317).
773
We measured the respiratory chain complex I, II⫹III,
and IV activities in a fresh muscle biopsy specimen and in
cultured fibroblasts according to standard procedures
(1 )(see the data supplement available with the online
version of this Technical Brief, at http://www.clinchem.
org/content/vol48/issue5/). In both samples we found
an isolated complex I deficiency (Table 1). The child died
at the age of 4 weeks from respiratory failure in the course
of an uncontrollable metabolic crisis.
At the time of the second pregnancy, the parents asked
for prenatal diagnosis. Because of the high variability of
the biochemical defect between the tissues, we try to
increase the diagnostic safety by offering prenatal diagnosis only if the biochemical defect is present in the index
patient’s muscle and cultured fibroblasts (2 ). To further
increase the safety margin, we measure respiratory chain
complex activities in native and cultured chorionic villi.
This policy was adopted after we had another patient
with complex I deficiency in whom the native chorionic
villi had normal activities, whereas the cultured ones
were clearly complex I-deficient. He died at 18 months
from severe lactic acidosis and hypertrophic cardiomyopathy (unpublished observation). In the second pregnancy
(II.2 in Fig. 1A), a chorionic villus biopsy was performed
at 11 weeks of gestation. We measured respiratory chain
enzyme activities in native as well as in cultured chorionic
villus biopsy specimens. In the native specimen, complex I
activity was normal, whereas it was pathologically decreased in the cultured specimen (Table 1). Because the
parents opted for a high degree of security, the decision
was made to terminate the pregnancy. Analysis of a
muscle specimen from the aborted female fetus, however,
revealed normal complex I activity.
At the time of the third pregnancy (II.3 in Fig. 1A), most
of the human respiratory complex I genes had been
cloned, and we were able to offer mutation screening. The
consanguinity of the parents led us to suspect a mutation
in one of the nuclear genes of complex I. We sequenced
the open reading frames of the NDUFV1, NDUFS4,
NDUFS7, and NDUFS8 genes, in which we had detected
mutations previously (3, 4 ). In the present case, we detected a homozygous C3 T transition at nucleotide 632 in
the patient’s NDUFV1 cDNA. The presence of the mutation was documented in genomic DNA by the gain of a
restriction site within the 230-bp PCR fragment generated
by the primer pair 5⬘-TgCAggTggCCATCCgAgAggCCTA-3⬘ (forward) and 5⬘-CACAgTCTgACCCagggTTACg-3⬘ (reverse), which was cleaved into 131- and 99-bp
fragments by Bsp1268I (Fig. 1B). The mutation was confirmed to be homozygous in the genomic DNA of the
index patient, whereas her parents and siblings were
heterozygous for this mutation. It was absent in 280 alleles
from healthy controls and in nearly 100 expressed sequence tags from GenBank. This excludes a common
polymorphism. The mutation changes an alanine to a
valine at amino acid 211 within the FMN-binding domain
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Technical Briefs
(5 ), which is strictly conserved down to Escherichia coli
(Fig. 1D). With 10 different mutations identified to date,
NDUFV1 seems to be a mutational hotspot in isolated
complex I deficiency (3, 6 ).
The finding of heterozygosity and normal complex I
activity in the muscular tissue of the aborted fetus (II.2)
was unexpected, based on the biochemical results of the
cultured chorionic villus specimen. Reanalysis of a second
cultured chorionic villus specimen stored in liquid nitrogen confirmed the original result. Unfortunately, no homogenate of the original native chorionic villi was available for reanalysis.
To resolve the discrepancy between the biochemical
results from native and cultured chorionic villi, we first
investigated the genetic identity of the cultured chorionic
villus specimen by analyzing nine different polymorphic
short tetranucleotide repeat loci and the X-Y homologous
gene amelogenin with the AmpFlSTRTM Profiler Plus Kit
(Applied Biosystems). The combination of these loci offers
an average probability of identity of P ⬍1.04 ⫻ 10⫺11 in
Caucasians. We found that the cultured chorionic villus
specimen was entirely of maternal, probably decidual,
origin (Fig. 1C). This had happened despite all precautions, including the commonly performed microscopic
check for maternal contamination. Milunsky and Cheney
(7 ) detected the presence of maternal cells in 3 of 24
chorionic villus samples and concluded that microscopic
examination of direct villi for apparent maternal cells is
not rigorous enough, especially in the face of very sensitive PCR techniques.
The finding of maternal cell overgrowth does not
explain the complex I deficiency of the cultured chorionic
villus specimen, which should be normal because the
mother is heterozygous for the mutation. Indeed, the
cultured fibroblasts of both heterozygous parents had
normal complex I activities (Table 1). We excluded a
chromosomal aberration in the cultured maternal cells (8 )
as a possible cause for the biochemical abnormalities. To
exclude a de novo mitochondrial DNA mutation as a
possible cause for the complex I deficiency, we additionally sequenced all mitochondrially encoded tRNAs and
ND genes of complex I in tissue DNA extracts from the
mother (I.1), the index patient (II.1), and cultured chorionic villi (II.2). In all samples we found the following
homoplasmic deviations from the standard GenBank sequence, NC_001807: MTND1, 3423T3 C (Val3 Val),
3480A3 G; MTND2, 4769A3 G; MTND3, 10398A3 G;
MTND4L, 10550A3 G, 10632T3 C (Leu3 Leu); MTND4,
11299T3 C, 11467A3 G, 11719G3 A; MTND5, 12372G3 A;
MTND6, 14167C3 T; and MTTL2, 12308A3 G. The underlined polymorphisms were not found in MITOMAP
(http://www.gen.emory.edu/mitomap.html) but did not
cause an amino acid exchange. All other polymorphisms
were already known and recorded in the MITOMAP
database.
We wished to determine whether the reference values
for decidual cells differed from those of chorionic villi. We
therefore prepared native decidual cells from healthy
placentas and cultured decidual cells from chorionic villus biopsy specimens that had been obtained for purposes
other than identifying mitochondrial diseases (see data
supplement). We measured the respiratory chain enzyme
activities in cultured as well as homogenized native
decidual cells. The complex I activity of the native chorionic villus sample was within the reference interval for
native decidual cells. The cultured decidual cells of the
second pregnancy, however, still showed a clear complex I
deficiency compared with control cultured decidual cells
(Table 1). These results did not place the complex I
activity of cultured cells from the second pregnancy
within the reference interval. Despite all efforts, we could
not find a satisfactory explanation for the results obtained
in the second pregnancy of this family.
Because the mutation was now known, at the time of
the third pregnancy we performed genetic and biochemical analyses with all available methods. This time the
Table 1. Respiratory chain enzyme activitiesa in fibroblasts, muscle tissue, chorionic villi, and decidual cells.
Source
Index patient (II.1)
Second pregnancy (II.2)
Third pregnancy (II.3)
Healthy controls (n ⫽ 5)
Healthy controls (n ⫽ 3)
Mother (I.1)
Father (I.2)
a
Material
Complex I
Complex IIⴙIII
Complex IV
Cultured fibroblastsb
Musclee
Native chorionic villie
Cultured decidual cellsb
Muscle (aborted fetus)e
Native chorionic villie
Cultured chorionic villib
Native decidual cellse
Cultured decidual cellsb
Cultured fibroblastsb
Cultured fibroblastsb
51 (100–260)c
24 (84–273)d
131 (84–263)d
120 (220–300)c
108 (84–273)d
160 (84–263)d
290 (220–330)c
122–414c
220–300a
275 (100–260)c
218 (100–260)c
234 (210–440)c
NDf
ND
270 (190–530)c
ND
ND
468 (190–530)c
ND
ND
941 (680–1190)d
1748 (520–2080)d
512 (271–922)d
830 (500–860)d
1119 (520–2080)d
550 (271–992)d
505 (500–860)d
122–208c
540–640c
The reference values are not normally distributed; therefore, the 5th to 95th centile range is indicated in parentheses after the measured values.
Activity measured in the mitochondrial enriched fraction.
c
Results are expressed as mU/U cytochrome c oxidase activity.
d
Results are expressed as mU/U citrate synthase activity.
e
Activity measured in the tissue homogenate.
f
ND, not determined.
b
Clinical Chemistry 48, No. 5, 2002
results were consistent and showed conclusively that (a)
both cultured and uncultured chorionic villi were of fetal
origin, (b) the 632C3 T transition was heterozygous (Fig.
1B), and (c) the complex I enzyme activities were normal
(Table 1). A healthy boy was born after an uncomplicated
pregnancy. He is now ⬎3 years of age and does not show
any symptoms of mitochondrial disease.
This case demonstrates the difficulties that can arise
from prenatal diagnosis based on biochemical results
alone. When results differ between native and cultured
cells, no safe prediction can be made. Unidentified culturing artifacts might influence the biochemical activity of
the cultured cells. Especially if the fetal cells are complex I
deficient, a few “healthy” maternal cells may overgrow
the fetal cells because of their advantage in oxidative
energy metabolism. When interpreting the results of biochemical tests on cultured chorionic villi, one should be
aware of the possibility of maternal cell contamination
and exclude it by microsatellite marker analysis. When
the fetus is male, a chromosome analysis might suffice.
We are grateful to the members of the family for participation in this study, to Dr. R. Knapp for providing the
patient material, to Dr. H. A. Zondervan and Vera Martens-Idenburg for providing the chorionic villi, to Christina Rovirosa and Loes van Moerkerken for technical
assistance, and to Dr. C. Gerstenfeld for critical discussions about the manuscript. This study was supported by
the Deutsche Forschungsgemeinschaft (SFB 577 TP B4)
and the parents’ support group “Helft dem muskelkranken Kind” (Hamburg).
References
1. Bentlage HA, Wendel U, Schägger H, ter Laak HJ, Janssen AJ, Trijbels JM.
Lethal infantile mitochondrial disease with isolated complex I deficiency in
fibroblasts but with combined complex I and IV deficiencies in muscle.
Neurology 1996;47:243– 8.
2. Ruitenbeek W, Sengers RCA, Trijbels JMF, Janssen AJ, Bakkeren JA. The use
of chorionic villi in prenatal diagnosis of mitochondriopathies. J Inherit
Metab Dis 1992;15:303– 6.
3. Schuelke M, Smeitink J, Mariman E, Loeffen J, Plecko B, Trijbels F, et al.
Mutant NDUFV1 subunit of mitochondrial complex I causes leukodystrophy
and myoclonic epilepsy. Nat Genet 1999;21:260 –1.
4. Smeitink J, van den Heuvel L, DiMauro S. The genetics and pathology of
oxidative phosphorylation. Nat Rev Genet 2001;2:342–52.
5. Fearnley IM, Walker JE. Conservation of sequences of subunits of mitochondrial complex I and their relationship with other proteins. Biochim Biophys
Acta 1992;1140:105–34.
6. Bénit P, Chretien D, Kadhom N, de Lonlay-Debeney P, Cormier-Daire V,
Cabral A, et al. Large-scale deletion and point mutations of the nuclear
NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency. Am J
Hum Genet 2001;68:1344 –52.
7. Milunsky JM, Cheney SM. Prenatal diagnosis of spinal muscular atrophy by
direct molecular analysis: efficacy and potential pitfalls. Genet Test 1999;
3:255– 8.
8. Sundberg K, Lundsteen C, Philip J. Comparison of cell cultures chromosome
quality and karyotypes obtained after chorionic villus sampling and early
amniocentesis with filter technique. Prenat Diagn 1999;19:12– 6.
775
Complete Sequencing of a Genetic Polymorphism in
NAT2 in the Korean Population, Soo-Youn Lee,1 Kyung-A
Lee,1 Chang-Seok Ki,1 O. Jung Kwon,2 Ho Joong Kim,2 Man
Pyo Chung,2 Gee Young Suh,2 and Jong-Won Kim1* (1 Department of Clinical Pathology and Division of Pulmonary
and Critical Medicine, 2 Department of Medicine,
Sungkyunkwan University School of Medicine and Samsung Medical Center, Seoul 135-710, Korea; * address
correspondence to this author at: No. 50, Ilwon-dong,
Kangnam-ku, Department of Clinical Pathology, Samsung Medical Center, Seoul 135-710, Korea; fax 82-2-34102719, e-mail [email protected])
N-Acetyltransferase 2 (NAT2) metabolizes arylamines
and hydrazines. The substrates of NAT2 include many
therapeutic drugs, such as isoniazid (INH), as well as
chemicals and carcinogens (1–3 ). For that reason, Nacetylation activity is associated with drug effects or
toxicities and susceptibility to various cancers. The ability
of NAT2 to N-acetylate arylamines is subject to a genetic
polymorphism in the NAT2 gene. The acetylation rate and
NAT2 genotype distribution are quite different among
various populations. The genetic polymorphism in NAT2
has not been studied extensively in the Korean population. Previous reports were based only on phenotyping or
restriction fragment length polymorphism analysis, leading to possible misclassification of genotypes. We therefore decided to investigate NAT2 allelic variability and
genotype distributions in the Korean population by complete sequencing. We also evaluated the relationship
between genotype and phenotype to understand N-acetylation pharmacogenetics.
One thousand Korean individuals who visited the
health promotion center at Samsung Medical Center were
anonymously studied. An additional 23 healthy volunteers and 18 patients with pulmonary tuberculosis participated in this study. DNA was extracted from peripheral
blood leukocytes. We amplified a 1211-bp fragment that
included an 870-bp protein-coding region of the NAT2
gene and performed full sequencing analysis (4 ) on an
ABI Prism 377 DNA Sequencer (Perkin-Elmer). We then
checked nucleotide substitutions by combined use of
allele-specific-PCR (AS-PCR) and restriction enzyme digestion. Specific primers (5 ) for the wild-type and mutant
alleles were used in separate PCRs to detect C282T,
T341C, and G590A substitutions. The nucleotides at positions 190, 481, 590, 803, and 857 were explored by digesting the 1211-bp PCR fragment or AS-PCR product carrying the wild-type or mutant allele with NciI, KpnI, TaqI,
DdeI, and BamHI, respectively. For phenotyping, the
healthy volunteers and patients gave informed consent
and were given 300 mg of INH orally after an overnight
fast. Participants were 18 – 65 years of age; had no underlying liver, kidney, or gastrointestinal diseases; did not
have any drug history within the previous 2 weeks; and
were not chronic alcoholics. Blood samples were obtained
at 6 h, and urine samples were collected up to 6 h after
INH administration. We measured urine and plasma
concentrations of INH and acetylisoniazid (AcINH) by