doi:10.1182/blood-2003-05-1537Prepublished online July 31, 2003;   

 NeufeldLaszlo G Boros, Mara P Steinkamp, Judith C Fleming, Wai-Nang Paul Lee, Marta Cascante and Ellis J thiamine-responsive megaloblastic anemia (TRMA)Defective RNA ribose synthesis in fibroblasts from patients with

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BLOOD (regular manuscript – accepted MS #2003-05- 1537)

Title: Defective RNA ribose synthesis in fibroblasts from patients with

thiamine-responsive megaloblastic anemia (TRMA)1.

Authors: László G. Boros1, Mara P. Steinkamp2, Judith C. Fleming2,4, Wai-Nang

Paul Lee1, Marta Cascante5 and Ellis J. Neufeld2,3,4

1Harbor-UCLA Research and Education Institute, UCLA School of Medicine, 1124 West

Carson street RB1, Torrance, CA 90502, USA; 2Division of Hematology/Oncology,

Children’s Hospital; 3Dana Farber Cancer Institute and 4Harvard Medical School 300

Longwood Avenue, Boston, MA 02115, USA; 5Department of Biochemistry and

Molecular Biology, University of Barcelona, Marti I Frankes 1, 08028 Barcelona,

Catalonia, Spain

Running Title: Thiamine transport and impaired nucleic acid synthesis

Keywords: pentose cycle, ribose synthesis, nucleic acid synthesis, thiamine transport; cell proliferation; stable isotope-based metabolic profiling (SIDMAP)

Address for correspondence: László G. Boros, MDCo-Director, Stable Isotope Research LaboratoryUCLA School of MedicineHarbor-UCLA Research and Education Institute1124 West Carson Street, RB1Torrance, CA 90502T: (310) 222-1856; Fax (310) 222-3887; E-mail: boros@gcrc.rei.edu

Word Count: Abstract: 200; Text: 3140

1 Supported in part by RO1 HL66182-01A1, K24 HLK24HL004184 & March of Dimes grants to EJN; PHS M01-RR0045 of the General Clinical Research Unit to WNPL; and P01-CA42710 of the UCLA Clinical Nutrition Research Unit Stable Isotope Core through its preliminary/feasibility grant program to LGB.

Copyright (c) 2003 American Society of Hematology

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Abstract

Fibroblasts from patients with thiamine-responsive megaloblastic anemia syndrome

with diabetes and deafness (TRMA) undergo apoptotic cell death in the absence of

supplemental thiamine in their cultures. The basis of megaloblastosis in these

patients has not been determined. Here we use the stable [1,2-13C2]glucose isotope-

based dynamic metabolic profiling technique to demonstrate that defective high-

affinity thiamine transport primarily affects the synthesis of nucleic acid ribose via

the non-oxidative branch of the pentose cycle. RNA ribose isolated from TRMA

fibroblasts in thiamine-depleted cultures shows a time-dependent decrease in the

fraction of ribose derived via transketolase, a thiamine-dependent enzyme in the

pentose cycle. The fractional rate of de novo ribose synthesis from glucose is

decreased several fold 2-4 days after removal of thiamine from the culture medium.

No such metabolic changes are observed in wild type fibroblasts or in TRMA

mutant cells in thiamine-containing medium. Fluxes through glycolysis are similar

in TRMA vs. control fibroblasts in the pentose and TCA cycles. We conclude that

reduced nucleic acid production through impaired transketolase catalysis is the

underlying biochemical disturbance which likely induces cell cycle arrest or

apoptosis in bone marrow cells and leads to the TRMA syndrome in patients with

defective high-affinity thiamine transport.

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Introduction

The pentose cycle, or “pentose-monophosphate shunt” allows de novo synthesis of ribose

from glucose by either or both of two possible routes: a “non-oxidative” pathway via

transaldolase and transketolase, and an “oxidative” pathway, via glucose 6-phosphate and

6 phosphogluconate dehydrogenases. The oxidative pathway also generates NADPH+

reducing equivalents. Whereas the oxidative branch of the pathway predominates in

circulating red blood cells, in many tissues the non-oxidative branch is quantitatively

more important for ribose synthesis destined for nucleic acids (1-3). Thiamine

pyrophosphate is a co-factor for transketolase, a key rate-limiting enzyme in the non-

oxidative pathway of the pentose cycle. Defective thiamine uptake in fibroblasts isolated

from patients with thiamine responsive megaloblastic anemia (TRMA) is associated with

the mutation and loss of function of the high-affinity, low-capacity thiamine transporter

(4). Fibroblasts isolated from patients with TRMA show cycle arrest and increased

apoptosis in low thiamine-containing media (5). Although thiamine deficiency has been

known to cause apoptosis of cells in sensitive brain regions including the thalamus,

mammillary and medial geniculate nuclei (6), the biochemical disturbance resulting from

defective high-affinity thiamine transport in human cells is yet to be elucidated.

Metabolic pathway flux changes appear to be critical factors in cell

differentiation, in apoptosis, and in mediating the anti-proliferative effect of imatinib

mesylate in Bcr-Abl positive myeloid leukemia cells in culture (7, 8). Stable isotope-

based dynamic metabolic profiling technology allows tracking of metabolic pathway

substrate flow changes in response to growth modulating treatments, or changes in the

nutrient environment (Figure 1) (9-11).

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Figure 1. The stable isotope-based dynamic metabolic profile (SIDMAP) of mammalian cells is based on 13C glucose carbon tracer rearrangements in metabolic intermediates. Schematic of pathways to ribose formation (1), glycolysis (2) and TCA cycle (3) from [1,2-13C2]glucose in cultured fibroblasts. This tracer broadly labels intermediary metabolites in cultured TRMA (-/-) cells that indicate substrate flow defects in comparison with WT (+/+) cells in low thiamine medium. Using SIDMAP, the rate of glycolysis, pentose cycle ribose synthesis, TCA cycle anaplerotic flux, direct glucose oxidation and de novo fatty acid synthesis are measured in one tracer experiment.

[1,2-13C2] Glucose 6-P

13C Fructose 6-P

13C Glyceraldehyde 3-P

13C labeled pentose productionRNA/DNA synthesisNADPH productionPentose recycling

13C labeled Lipids

Krebs cycle

13C labeled amino acids

αααα-ketoglutarate13C glutamate

13C Pyruvate - Lactate

citric acid

13C oxaloacetate

[1,2-13C2]glucose tracer Glycogen

Acetyl-CoA2

1

3

Protein production

Plasma membrane

13C Glucose 1-P

Here we demonstrate, using stable isotope-labeled glucose and its recovered

intracellular metabolites, that the defective high-affinity thiamine transporter protein

produced by the mutated SLC19A2 gene in TRMA impairs nucleic acid synthesis in the

non-oxidative branch of the pentose cycle. The mutant protein also produces smaller,

non cell-specific effects on glycolysis, the oxidative pentose cycle, and distribution of

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glucose carbons for de novo fatty acid synthesis or glucose anabolic use in the TCA

cycle. These observations suggest a possible novel etiologic mechanism for

megalobastosis: namely, defective re-distribution of glucose carbons among

macromolecule biosynthesis pathways in affected precursor marrow cells of patients with

TRMA.

Materials & Methods

The tracer for this metabolic profiling study, stable isotope [1,2-13C2]- labeled D-

glucose, was purchased with >99% purity and 99% isotope enrichment for each position

from Isotec Laboratories, Inc., Maimisburg, OH.

Cells & cell culture

Human fibroblasts were obtained from skin biopsies of patients and unaffected

relatives of two previously reported TRMA kindreds, after obtaining informed consent

from subjects(12, 13). Cells were grown in minimum essential medium (MEM) in the

presence of 15% FBS, at 37oC in a 95% air-5% CO2. In order to compare glucose

utilization rates, ribose synthesis, lactate production, fatty acid de novo synthesis and

anabolic glucose use in the TCA cycle, 75% confluent cultures of control and TRMA cell

lines were incubated in [1,2-13C2]D-glucose-containing media (100 mg/dl total

concentration = 5 mM; 50% isotope enrichment – i.e. half unlabeled glucose, half labeled

with the stable isotope 13C tracer). Cells were plated at a density of 106 per 100 mm2

dish. Medium depleted of thiamine (thi-) and dialyzed FBS were obtained from

GIBCO/BRL as previously described (12). Medium glucose and lactate levels were

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measured using a Cobas Mira chemistry analyzer (Roche Diagnostics, Pleasanton, CA,

USA). Fibroblast metabolic profiles and macromolecule synthesis patterns were

determined at days 2, 4 and 7 in order to reveal time dependence of thiamine depletion in

cultured cells up to the time when apoptosis occurs in TRMA cells.

RNA ribose stable isotope studies

RNA ribose was isolated by acid hydrolysis of cellular RNA after Trizol

purification of cell extracts. Total RNA amounts were assessed by spectrophotometric

determination on triplicate cultures. Ribose was derivatized to its aldonitrile acetate form

using hydroxylamine in pyridine with acetic anhydride (Supelco, Bellefonte, PA) before

mass spectral analyses. We monitored the ion cluster around the m/z256 (carbons 1-5 of

ribose) (chemical ionization, CI) and m/z217 (carbons 3-5 of ribose) and m/z242 (carbons

1-4 of ribose) (electron impact ionization, EI) to determine molar enrichment and the

positional distribution of 13C in ribose. By convention, the base mass of 12C- compounds

(with their deriviatization agents) is given as m0 as measured by mass spectrometry.

Ribose molecules labeled with a single 13C atom on the first carbon position (m1)

recovered from RNA were used to gauge the ribose fraction produced by direct oxidation

of glucose through the G6PD pathway (Figure 1). Ribose molecules labeled with 13 C on

the first two carbon positions (m2) were used to measure the fraction produced by

transketolase. Doubly labeled ribose molecules (m2 and m4) on the fourth and fifth

carbon positions were used to measure molar fraction produced by triose phosphate

isomerase and transketolase (3, 10, 11).

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Lactate

Lactate from the cell culture media (0.2 ml) was extracted by ethylene chloride

after acidification with HCL. Lactate was derivatized to its propylamine-HFB form and

the m/z328 (carbons 1-3 of lactate) (chemical ionization, CI) was monitored for the

detection of m1 (recycled lactate through the PC) and m2 (lactate produced by the

Embden-Meyerhof-Parnas pathway) for the estimation of pentose cycle activity.

Fragmental lactate analysis was not necessary because the [1,2-13C2]glucose tracer labels

lactate on the 3rd (m1) or the 2nd and 3rd (m2) carbon positions, which are clearly

distinguished in the molecular ion (3). In this study we recorded the m1/m2 ratios in

lactate produced and released by TRMA and control fibroblasts in order to determine

pentose cycle activity versus glycolysis in response to thiamine depletion.

Glutamate

The TCA cycle metabolite alpha-ketoglutarate is in close equilibrium with

glutamate, which is released by mammalian cells into the medium. Glutamate label

distribution from glucose is suitable for determining glucose oxidation versus anabolic

glucose use within the cycle, also known as anaplerotic flux (15). In order to obtain

glutamate, tissue culture medium was first treated with 6% perchloric acid. The

supernatant was passed through a 3 cm3 Dowex-50 (H+) column. Amino acids were

eluted with 15 ml 2N ammonium hydroxide. To further separate glutamate from

glutamine, the amino acid mixture was passed through a 3 cm3 Dowex-1 (acetate) column

then collected with 15 ml 0.5 N acetic acid. The dry glutamate fraction from tissue

culture medium was converted to its trifluoroacetyl butyl ester (TAB) (16). Under EI

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conditions, ionization of TAB-glutamate gives rise to two fragments, m/z198 and m/z152,

corresponding to C2-C5 and C2-C4 of glutamate. Glutamate labeled on the 4-5 carbon

positions indicates pyruvate dehydrogenase activity while glutamate labeled on the 2-3

carbon positions indicates pyruvate carboxylase activity for the entry of glucose carbons

to the TCA cycle. TCA cycle anabolic glucose utilization is calculated based on the

m1/m2 ratios of glutamate (15).

Fatty acids

Palmitate, stearate, cholesterol and oleate were extracted after saponification of

cell pellets in 30% KOH and 100% ethanol using petroleum ether. Fatty acids were

converted to their methylated derivative using 0.5N methanolic-HCL. Palmitate, stearate

and oleate were monitored at m/z270, m/z 298 and m/z264, respectively, with the

enrichment of 13C labeled acetyl units which reflect synthesis, elongation and

desaturation of the new lipid fraction in fibroblasts in response to thiamine depletion in

the medium as determined by mass isotopomer distribution analysis (MIDA) of different

isotopomers (17).

Gas Chromatography/Mass Spectrometry (GC/MS)

Mass spectral data were obtained on the HP5973 mass selective detector

connected to an HP6890 gas chromatograph. The settings were as follows: GC inlet

250oC, transfer line 280oC, MS source 230oC, MS Quad 150oC. An HP-5 capillary

column (30m length, 250µm diameter, 0.25µm film thickness) was used for glucose,

ribose and lactate analyses. Because transketolase has the highest metabolic control

coefficient in the non-oxidative branch of the pentose cycle and it is the only thiamine-

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dependent reaction associated with this cycle (19, 20), we use the terms non-oxidative

pentose cycle and transketolase interchangeably throughout the paper. It should be noted

though that transketolase and transaldolase, besides other enzymes, are all participants of

non-oxidative metabolism in human cells.

Data analysis and statistical methods

Each experiment was carried out at least twice for every condition, and triplicate

cell cultures were used for each condition within each experiment. Mass spectroscopic

analyses were carried out by three independent automatic injections of 1 µl samples by

the automatic sampler and accepted only if the standard sample deviation was less than

1% of the normalized peak intensity. Statistical analysis was performed using the

Student’s t-test for unpaired samples. Two-tailed significance at the 99% confidence

interval (µ+/-2.58σ), p<0.01 indicated significant differences in glucose carbon

metabolism in wild type and TRMA cells in culture at various time points of thiamine

depletion.

Results

The SIDMAP approach has been successful in determining pentose cycle activity

and the contribution of the two branches of the pentose cycle to nucleic acid synthesis in

HepG2 liver (18), MIA pancreatic adenocarcinoma (21), K562 myeloid leukemia cells (7,

8) in previous studies. In this study, we incubated human wild type and TRMA

fibroblasts with [1,2-13C2]glucose in the presence or absence of thiamine in order to

determine 13C label distribution in RNA ribose, lactate and glutamate, and to determine

the contribution of glucose carbons to de novo nucleic and fatty acid synthesis. Based on

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the number of 13C labeled carbons present in the extracted small molecular weight

intermediary metabolites, the percent of metabolite synthesis via each pathway was

determined. Net de novo RNA synthesis was dramatically reduced in thiamine-depleted

TRMA cells, compared to thiamine-replete mutant cells or to wild type control cells

(Figure 2).

Figure 2. TRMA cells without thiamine exhibit decreased RNA ribose synthesis via transketolase. The fraction of RNA ribose derived from the oxidative (G6PD) and non-oxidative (TK) pathways is shown on “X”. The figure shows the significant rearrangement of oxidative/nonoxidative ribose synthesis pathways in TRMA cells, primarily shifting toward oxidative synthesis, while wild type cells maintain metabolic activities and identical substrate flow in the pentose cycle after thiamine depletion. (Mean +/- SD; n=9) *p<.001

+ thiamine - thiamine0

10

20

30

40

50

60

70

80

90

100TRMA fibroblasts

G6PDH ribose TK ribose

+ thiamine - thiamine0

10

20

30

40

50

60

70

80

90

100wt fibroblasts

Fra

ctio

n o

f R

NA

rib

ose

(%

of

tota

l)

*

*

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This analysis depends on the fact that net increases in 13C ribose can only occur

by de novo ribose synthesis from [1,2 13C2]D-glucose. Change in the route of de novo

ribose synthesis was evident after two days of culture. In the presence of thiamine, both

the wild type fibroblasts and the mutated cells synthesize ~80% of nucleic acid ribose

directly through the non-oxidative branch and ~20% directly through the oxidative

G6PDH reaction. Control normal fibroblasts showed no change in the contribution of

transketolase and G6PD to nucleic acid ribose under thiamine-depleted conditions.

TRMA fibroblasts, on the other hand, showed a significant 25% decrease in the

contribution of transketolase and a corresponding 37% increase in the contribution of

direct glucose oxidation through G6PD to nucleic acid ribose synthesis after two days of

culture under thiamine-depleted conditions (Figure 2).

The impact of time on metabolic changes in TRMA cells in the absence of

thiamine was also evident. A time-dependent decrease in the contribution of

transketolase and a compensatory increase in the contribution of G6PDH to nucleic acid

ribose synthesis was evident in TRMA fibroblasts incubated in thiamine-depleted

medium with [1,2-13C2]glucose for 2, 4 and 7 days, respectively (Figure 3). Signs of

apoptotic cell death accompanied the prominent decrease in the contribution of

transketolase to nucleic acid ribose synthesis in TRMA cells at day 7 of thiamine

depletion, similar to previous observations (12). This metabolic change was absent in

thiamine-replete cultures where apoptosis did not occur, indicating that adequate

thiamine supplementation is capable of maintaining non-oxidative ribose synthesis in

TRMA cells and maintaining phenotype.

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Figure 3. TRMA cells without thiamine exhibit a time-dependent decrease in RNA ribose synthesis via transketolase. There is a time-dependent decrease in de novo RNA ribose synthesis through the nonoxidative pathway in TRMA cells depleted of thiamine and there is a compensatory increase in glucose oxidation and ribose synthesis though G6PDH. Over seven days, the fraction of RNA ribose derived from the oxidative pathway under thiamine-depleted conditions is approximately twice that in thiamine-replete medium. WT cells did not exhibit significant change in pentose cycle carbon flow between glucose oxidation and non-oxidative ribose synthesis at any time point throughout our study. (Mean +/- SD; n=9) *p<.001

0

20

40

60

80

100

Transketolase G6PD

WT cells TRMA cells WT cells TRMA cells

Pro

po

rtio

nal

rib

ose

syn

thes

is f

or

RN

A (

% o

f to

tal)

Da y

2

Day

2D

ay 4

Day

7

Day

4D

ay 7

Day

7

Day

7

Da y

4D

a y 2

Day

4D

ay 2

*

**

*

**

The sum of all labeled species, (Σmn) found in RNA ribose is a measure of de

novo synthesis from [1,2-13C2]D-glucose. Total ribose synthesis has also significantly

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declined in TRMA fibroblasts as shown in figure 4. The rate of de novo incorporation

was measured by the change (∆) in 13C enrichment total RNA. There is a significant

decrease of about 80% in TRMA cell total RNA synthesis from glucose after 2 days of

thiamine depletion, a phenomenon that has not been observed in wild type fibroblasts.

Figure 4. TRMA cells without thiamine exhibit a significant decrease in total RNA ribose synthesis after 2 days of thiamine depletion. There is a significant decrease of about 80% in TRMA cell total RNA synthesis from glucose after thiamine depletion, which has not been observed in wild type fibroblasts. (Mean +/- SD; n=9) *p<.001

WT TRMA0.00

0.04

0.08

0.12

0.16

0.20

∆(∆( ∆(∆(fr

acti

on

al r

ibo

se la

bel

)

+ th

iam

ine

+ th

iam

ine-th

iam

ine

-th

iam

ine

Pentose cycle activity relative to the Embden-Meyerhoff-Parnas pathway was

calculated by the m2/m1 ratios measured in lactate using previously published methods

*

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and formulas (3). Pentose cycle activity showed a modest time-dependent increase in

both thiamine depleted and thiamine-replete TRMA cultures over time. This change did

not differ with thiamine states: note that only ~2% of lactate is derived via the pentose

cycle, while the remainder is derived from glycolysis in TRMA cells (Figure 5).

Figure 5. Slight time dependent increase in pentose cycle activity relative to glycolysis in TRMA cells with or without thiamine treatment. Pentose cycle activity relative to the Embden-Meyerhoff-Parnas pathway is shown here by the m1/m2

ratios in lactate. Pentose cycle activity showed a modest time-dependent increase in both thiamine depleted and thiamine-replete TRMA cultures. (Mean +/- SD; n=9) *p<.001

0

0.5

1

1.5

2

2.5

3

1

Day

2

Day

7

Day

4

Day

2

Day

4

Day

7

Pen

tose

cyc

le a

ctiv

ity

( % o

f gl

y col

ysi s

)

(+) Thiamine (-) Thiamine

Total mass and fragmentation patterns in glutamate are unique and depend

primarily on pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (α-

KTGDH) activities as the thiamine dependent steps in the TCA cycle. The position of

the 13C label in glutamate (either C 2-3 or C 4-5) allows the estimation of TCA cycle

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anaplerotic flux relative to glucose oxidation. As shown in Figure 6 depletion of

thiamine from the culture media of TRMA cells induce a significant increase in TCA

cycle anaplerotic flux, indicating that mutation in the high affinity thiamine transporter

gene does not decrease TCA cycle anaplerotic flux and carbon flow.

Figure 6: TCA cycle anaplerotic flux increases in TRMA cells as measured by glutamate 13C enrichment from glucose. As shown here, depletion of thiamine from the culture media of TRMA cells induces a significant increase in TCA cycle anaplerotic flux. (Mean +/- SD; n=9) *p<.001

0

0.8

1.6

2

1

Day

2

Day

4

Day

7

(+) Thiamine (-) Thiamine

* *

TC

A c

ycle

ana

pler

otic

flux

(%

of

gluc

ose

oxid

atio

n)

Day

2

Day

4

Day

7

Control wild type cells showed no response in TCA cycle metabolism in response to

thiamine depletion in the medium (not shown). Based on this finding, the only thiamine

dependent enzyme that also depends on the high-affinity transporter in TRMA cells is

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transketolase, an interrelationship demonstrated by the decrease in non-oxidative pentose

cycle metabolism shown above (Figure 3).

Significant accumulation of 13C tracer from glucose did not occur in the fatty

acids palmitate, stearate and oleate, or in cholesterol, indicating that fatty acid de novo

synthesis, chain elongation and desaturation is not very active in control and TRMA

cells. This confirms that the thiamine-PP dependent pyruvate dehydrogenase enzyme

complex is not affected by the defective high-affinity thiamine transporter in TRMA

fibroblasts (data not shown).

Discussion

We have used stable isotope-based dynamic metabolic profiling to demonstrate

defective de novo nucleic acid synthesis in TRMA fibroblasts subjected to thiamine

deprivation. The fractional synthetic rate is reduced in thiamine depleted TRMA cells.

In addition, the fraction of ribose derived from the preferred non-oxidative

(transketolase/transaldolase) portion of the pentose phosphate pathway is reduced in

favor of the oxidative pentose pathway branch (via G6PDH). A comparison of

SIDMAPs of TRMA (-/-) cells in thiamine-PP supplied versus depleted conditions is

shown in figure 7.

Figure 7. Metabolic profile changes associated with thiamine-PP depletion in TRMA (-/-) cultures exhibiting apoptosis. TRMA cells in the presence of thiamine utilize glucose as the major substrate for de novo nucleic acid synthesis through the non-oxidative steps of the pentose cycle (left). Non-oxidative ribose synthesis decreases while direct glucose oxidation increases in the pentose cycle(right). TCA cycle substrate flux showed no significant response to thiamine depletion indicating well preserved pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase activities as other thiamin dependent enzymes of metabolism. SIDMAP provides a virtual roadmap by which regulation of cell

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cycle, enzyme protein synthesis as well as gene expression can be pursued for revealing the mechanism of TRMA cells’ metabolic deficiencies.

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl -CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl-CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl-CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

NADPHGlucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl -CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl-CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl-CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl -CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl-CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl-CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl-CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

TRMA (-/-) CELLS TRMA (-/-) CELLS

THIAMINE SUPPLIED CONDITION THIAMINE SUPPLIED CONDITION THIAMINE SUPPLIED CONDITION THIAMINE SUPPLIED CONDITION THIAMINE DEPLETED CONDITIONTHIAMINE DEPLETED CONDITIONTHIAMINE DEPLETED CONDITIONTHIAMINE DEPLETED CONDITIONGlucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl -CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl -CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

NADPHNADPH

Glucose

G6P

F6P

GAP

PYRLACTATE

???? -ketoglutarate

OAA Acetyl-CoA

GLUTAMATE

RIBOSE

FATTY ACIDS

PDH PDH PDH PDH PDH PDH PDH PDH

TRANSKETOLASE TRANSKETOLASE TRANSKETOLASE TRANSKETOLASE TRANSKETOLASE TRANSKETOLASE TRANSKETOLASE TRANSKETOLASE

G6PDH G6PDH G6PDH G6PDH G6PDH G6PDH G6PDH G6PDH

Apoptosis/death

Survival/proliferation

What are the likely consequences of this change?

TRMA fibroblasts undergo apoptosis in thiamine-depleted conditions after about

two weeks in culture (5). It is evident that the observed decreases in nucleic acid

synthesis significantly contribute to this effect. In TRMA patients, the ambient thiamine

concentration is not zero. However, hematopoietic cells in the marrow require very high

nucleic acid synthesis rates to achieve division times which are very much greater than

those of leisurely diploid fibroblasts in culture (doubling time << 1 day vs. several days).

Drugs (e.g. folate antagonists or purine analogs), metabolic disorders (orotic aciduria,

Lesch-Nyhan syndrome) and nutritional states (B12 or folate deficiency), which reduce

de novo nucleotide synthesis, lead to a “megaloblastic” appearance of the marrow, with

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nuclear-cytoplasmic dyssynchrony and delayed nuclear maturation. We postulate the

same effect in TRMA erythropoiesis with the following notions. Since the initial report

of TRMA in 1969 by Rogers and colleagues (22), it has been clear that the megaloblastic

changes in this disorder differ from other vitamin deficiency states. High dose B12 and

folate have no effect on the condition. Haworth, et al performed deoxyuridine

suppression tests on marrow from a Pakistani child with TRMA and found no defect (23).

Deoxyuridine fails to inhibit incorporation of [3H]thymidine into DNA if it cannot be

converted (via folate- dependent uridine methyltransferase) into TMP. This step is

unaffected in TRMA fibroblasts. The high affinity thiamine transporter defective in

TRMA, designated SLC19A2, does not transport folate (24), though a report suggests

that the homologue and reduced folate carrier, SLC19A1, could carry thiamine

pyrophosphate in an experimental system (25). The SLC19A1 gene is not defective in

TRMA, so that to the extent this observation is correct, it does not rescue TRMA

fibroblasts from the biochemical disturbance noted here.

We acknowledge two potential limitations of this study. First, because fibroblasts

grow very slowly and do not have the specialized requirement of erythropoietic

precursors for rapid growth and hemoglobin synthesis, we cannot directly test the

hypothesis about megaloblastic changes in these cells alone. Conditions to test thiamine

metabolic effects in marrow are under development, but these studies are not

straightforward. In any case, marrow from TRMA patients is not readily obtainable and

patients are universally thiamine-treated. Second, the GC-MS analyses are highly

accurate and reproducible with regard to molar fractions with varying label

concentrations, but not well suited to accurate mass calculations without a tracer.

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Fortunately, as the labeled [1,2 13C2]glucose is initially incorporated into RNA, the

change in molar enrichment directly reflects the de novo synthesis rate (as shown in

figure 1). We also experimented using 13C ribose tracer in TRMA fibroblast cultures, but

these cells did not accumulate measurable tracer-ribose, probably due to the lack of

ribose transport in human cells. Human cells primarily utilize glucose for ribose

synthesis and this makes glucose tracers superior for SIDMAP studies as presented

herein.

Whereas hematologists are used to thinking of the pentose phosphate “shunt” as

an erythrocyte pathway for generation of reducing equivalents, most mammalian cell

types preferentially use the non-oxidative direction through the pentose phosphate

pathway to generate net pentose synthesis (26). Reduced NADH and NADPH can be

generated by mitochondrial or lipolytic activities in most cell types, but not in

erythrocytes. The observation is not new that the transketolase (non-oxidative) pathway

is preferred in cultured cells and organs, having first been made more than 40 years ago

by Hyatt and by Horecker, using isotopic enrichment experiments similar in concept to

those described here (1, 2).

Based on these data, the predominant metabolic disturbance in TRMA patients is

decreased nucleic acid ribose synthesis through the non-oxidative branch of the pentose

cycle (Figure 7). The prominent decrease observed in the contribution of transketolase to

nucleic acid ribose synthesis indicates that thiamine-PP and non-oxidative ribose

synthesis are required for human cell survival and proliferation. This observation may

also identify a new enzymatic target site in the apoptotic pathway of mammalian cells,

which is regulated primarily through high-affinity thiamine transport. Transaldolase, a

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non-oxidative pentose cycle enzyme, has been shown to regulate the metabolic flux

between the oxidative and non-oxidative branches of the pentose cycle and to influence

sensitivity to various cell death signals (27). Transketolase has a higher metabolic

control coefficient in the pentose cycle, which makes it a close rate-limiting enzyme in

non-oxidative ribose synthesis (19). Therefore it is not surprising that human cells are

sensitive to thiamine depletion and reduced transketolase activity when their high affinity

thiamine transport fails. One interesting aspect of our study is that the data suggest that

high-affinity low capacity thiamine transport may be linked to transketolase-related

metabolic activities while TCA cycle metabolism, glycolysis and fatty acid synthesis

pathways are not affected. Therefore, it seems likely that high affinity thiamine transport

is tied to nucleic acid synthesis and cell replication as a primary and highly important

mammalian cell function for survival and progression. Both the fractional rate of label

incorporation and the route of label within the pentose cycle are abnormal in TRMA

cells. This finding suggests that defective ribose synthesis for nucleic acids is the likely

cause of megaloblastic changes in TRMA.

Acknowledgements: We are grateful to the patients and their families for

participating in this study.

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