Bernard R. Landau,* John Wahren,i Visvanathan Chandramouli,* William C. Schumann,* Karin Ekberg,i
and Satish C. Kalhan
Departments of *Medicine, Biochemistry and Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106;
and iDepartment of Clinical Physiology, Karolinska Hospital, S 171 76 Stockholm, Sweden
of 0.1 N H2SO4 was placed in a boiling water bath for 1 h to hydrolyze moval of the nucleotides from solution was followed by the disap-
the monoacetone xylose to xylose. The hydrolysate was passed pearance of absorption at 260 mu. The supernatant was extracted
through a column of 1.5 grams AG 50W-X8 in the hydrogen form with ether (2 ml 3 3) to remove trichloroacetic acid. It was then
over 1.5 grams AG 1-X8 in the formate form (Bio-Rad Labs, Her- passed through the AG 1-X8 ion exchange column in the formate
cules, CA). The column was washed with water and the effluent evap- form. The column was washed with water, and then the ribulose-5-P
orated to dryness. was eluted with 1 N formic acid. That fraction was evaporated to dry-
The xylose was purified by HPLC using a Bio-Rad HPX-87P col- ness.
umn with water as solvent at 808C and a flow rate of 0.5 ml/min. Xy- When (2-3H, 1-14C)glucose was carried through to this stage of the
lose eluted at 1618 min. The amount of xylose in the eluate was procedure z 60% of the 3H in the glucose was recovered in the 1 N
determined by the Dische method (6). The yield from glucose was formic acid fraction. The amount of ketopentose in the fraction, de-
4045% of theoretical. A similar yield was obtained beginning with termined using the Dische colorimetric assay (6), was also z 60% of
2 mg of glucose. An adequate yield of xylose for preparation of HMT theoretical. After treatment with phosphatase, on HPLC using the
has been obtained beginning with only 0.7 mg of glucose. In accord HPX-87P column, there was a single radioactive peak with the mobil-
with the removal of carbon 6, when (6-3H, 1-14C)glucose was con- ity of ribulose, giving the color described for a ketopentose (6). While
verted to xylose, 99% of the 3H was lost relative to 14C, and when an HMT the amount of ribose-5-P isomerase in the enzyme preparation used
was prepared from xylose from (6-3H, 5-14C)glucose . 99% of the 3H was reported to be , 0.03%, we were concerned about ribose-5-P as
was lost relative to 14C and from (5-3H, 6-14C)glucose 98% of the 14C was a contaminant. However, there was no evidence of contamination on
lost relative to 3H. The 3H/14C ratio in the HMT from (5-3H,5- HPLC, the hydrogen lost in the isomerization of ribulose-5-P to ri-
14
C)glucose was the same as in the glucose, indicating no loss of the bose-5-P is not the hydrogen bound to carbon 2 of the glucose, and
hydrogen and no isotope effect. The (1-14C)glucose, (6-14C)glucose, reduction of ribose-5-P also yields ribitol-5-P.
(5-3H)glucose, and (6-3H)glucose were purchased commercially and The ribulose-5-P was dissolved in 0.3 ml of water, the solution
the (5-14C)glucose was synthesized (7). taken to pH 7 with NaOH, and 2 mg of NaBH4 was added. After 12 h
Glucose conversion to ribitol-5-P and arabitol-5-P (Fig. 3). The con- at room temperature 0.08 ml of 2 N acetic acid followed by 0.05 ml of
version of glucose to ribulose-5-P was essentially as described (8, 9). 7 M NH4OH was added, giving a pH of 89. The solution was diluted
The reactions were taken to completion by coupling them to the con- to 2 ml with water and passed through a 1 ml column of the AG 1-X8
version of a-ketoglutarate to glutamate. The reduction of the ribu- resin in the acetate form. The column was washed with water and
lose-5-P to the polyol phosphates was done by a microscale modifica- then the polyol phosphates eluted in about 2 ml of 1 N ammonium ac-
tion of the method described in references 10 and 11 (Fig. 3). 2 mg of etate. Cations were removed from the eluate by adding 23 grams of
glucose was incubated for 1 h at 308C in 1 ml of 0.1 M triethanolamine AG-50W in the H1 form. The supernatant combined with water
buffer, pH 7.6, with 10 mM MgCl2, 8 mg of ATP, and 4 U of hexoki- washings of the resin was evaporated at room temperature with addi-
nase. Conversion of the glucose to glucose-6-P was virtually complete tions of water to remove acetic acid, and then evaporated to dryness.
as evidenced by retention on an anion exchange column of all the 14C Traces of borate were removed by addition and evaporation of meth-
when an incubate in which the glucose had been labeled with (1-14C) anol (2 ml 3 3).
glucose was passed through the column. After 1 h, 5 mg of a-keto- The yield of polyol phosphates from the glucose was z 30% of
glutarate, 2 mg of NH4Cl, 6 mg of NADP, 1 U of glucose-6-P dehy- theoretical. That yield is based on recovery of 3H beginning with
drogenase, 1 U of 6 phosphogluconate dehydrogenase, and 1.5 U of (2-3H)glucose. After phosphatase treatment, two peaks with about
glutamate dehydrogenase were added, giving a total volume of incu- equal radioactivity were found on HPLC using the HPX-87P column,
bate of 1.2 ml (all enzymes and nucleotides were purchased from one with the mobility of ribitol and the other with that of arabitol. An
Sigma Chemical Co., St. Louis, MO). The pH was adjusted to 7.7, re- HMT prepared from the polyol phosphates obtained on incubation of
quiring a small amount, z 0.1 ml, of 1 N NaOH and the incubation (2-3H, 2-14C)glucose had the same 3H/14C ratio as the glucose. When
continued for an additional 3 h at 308C. (2-3H, 1-14C)glucose was incubated, HMTs had , 2% of the 14C rela-
To stop the reaction 0.1 ml of 50% trichloroacetic acid was added tive to 3H in the glucose, in accord with the removal of carbon 1. An
and then a pinch of activated charcoal to absorb the nucleotides (12). HMT, when (2-3H, 6-14C)glucose was incubated, had , 0.5% of 14C
The mixture was centrifuged, another pinch of the charcoal added to relative 3H in the glucose, in accord with no formaldehyde being
the supernatant, and the resulting mixture again centrifuged. The re- formed on periodic acid oxidation from position 6 of glucose when
Subject Time Carbon 2 (42 h) Urinary water Carbon 2 (42 h) Carbon 2 (42 h)
then bear no 2H at carbons 2, thus resulting in an underestima- between carbons 1 and 2 of the hexose-6-phosphates (16). In-
tion of the contribution of glycogenolysis. Correcting for that tramolecular movement of the hydrogen bound to carbon 2 of
glucose increases the contribution of glycogenolysis at 14 h glucose-6-P formed in glycogenolysis began with a hydrogen
from 51% to [(51 1 5)/105]100 5 53% with the contribution of unenriched in deuterium. However, movement of the hydro-
gluconeogenesis reduced to 4764%. The corrected contribu- gen bound to carbon 1 of fructose-6-P formed by gluconeogen-
tions of gluconeogenesis are then 6764% at 22 h and 9362% esis began with a hydrogen already enriched to the extent of
at 42 h. the hydrogens bound to carbon 6. That is because the four hy-
The enrichment in urinary water collected at 1418 h was drogens bound to carbons 1 and 6 of the fructose-6-P derive
the same, z 0.5%, as at 3842 h, as expected from the amounts from the hydrogens bound to carbon 3 of the triose phos-
and times of 2H2O administrations. That agrees with our previ- phates. That the hydrogen bound to carbon 2 of glucose-6-P
ous experience under the same conditions. Thus, a constant formed from fructose-6-P by gluconeogenesis reached the en-
enrichment in urinary water was achieved by 14 h and presum- richment in body water is evidenced by the absence of a dis-
ably for several hours before, since the last dose of 2H2O was cernible difference between the enrichment at carbon 2 at 42 h
ingested 9 h into the fast. and the enrichment in urinary water.
In the conversion of fructose-6-P to glucose-6-P during glu- The lower ratio of enrichment at carbon 2 at 14 and 22 h to
coneogenesis, there is labeling at carbon 2 of the glucose-6-P that at 42 h then provides a measure of the extent of the equil-
through an exchange with water. Exchange resulting in labeling ibration of the hydrogen at carbon 2 of glucose-6-P with that in
at carbon 2 of glucose-6-P formed by glycogenolysis depends water during glycogenolysis. Since the mean ratio at 1442 h
upon the extent the glucose-6-P is converted to fructose-6-P was 0.89 when there was 53% glycogenolysis, the extent of
before its conversion to glucose. When (2-3H, 2-14C)galactose equilibration was [(89 2 47)/53]100 5 79%. At 22 h it was [(95 2
was infused into subjects fasted overnight, the 3H/14C ratio in 67)/33]100 5 85%. We do not know why we did not find lower
blood glucose was z 20% of that in the galactose, suggesting enrichments at carbon 2 at 14 h than at 42 h in our previous
that in gluconeogenesis z 80% of the hydrogen at carbon 2 study (1), when assaying enrichment by transferring the hydro-
equilibrates with water (2). In addition to exchange with water, gen at carbon 2 to lactate.
interconversion of glucose-6-P with fructose-6-P occurs with The proportions of gluconeogenesis calculated from the ra-
hydrogen transfer, the intramolecular movement of hydrogen tios at carbon 6 to carbon 2 at 42 h were 3363% at 14 h,