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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 691-694
Fetal Hemoglobin in Starvation Ketosis of Young Women
By
Achim Peters,
Dagmar Rohloff,
Thomas Kohlmann,
Florian Renner,
Günther Jantschek,
Wolfgang Kerner, and
Horst Lorenz Fehm
From the Medical Clinic 1, the Institute for Social Medicine, the
Institut for Clinical Chemistry, and the Medical Clinic 2, Medical
University of Lübeck, Lübeck, Germany; and the Department
of Diabetes and Metabolic Disorders, Klinikum Karlsburg, Karlsburg,
Germany.
 |
ABSTRACT |
Ketones can reactivate the production of fetal hemoglobin (HbF) in
vitro and in vivo. A reactivation of HbF by ketones, which are
generated during starvation, remains largely speculative. Therefore, we
investigated HbF in 31 women with anorexia nervosa or bulimia, using
both of these as models of intermittent starvation ketosis. For comparison, we also studied 42 female control
subjects matched for age.  -Hydroxybutyrate levels were
higher in patients than in controls (460 ± 90 v 110 ± 20 µmol/L; P < .0001). We correlated -hydroxybutyrate,
metabolic, and hematologic parameters with HbF. HbF was measured with
high pressure liquid chromatography. The data were analyzed with
logistic regression analysis. An elevated HbF fraction (>0.87%) was
observed four times as often in patients than in controls (29%
v 7%, P = .01). After adjustment for age, we found
HbF elevations associated with -hydroxybutyrate levels (P
= .005). No other correlations between the various
metabolic/hematologic parameters and HbF were significant. In
conclusion, -hydroxybutyrate generated in starvation is associated
with increased levels of HbF. Thus, unrestrained lipolysis can produce
-hydroxybutyrate in sufficient quantities to induce a clinically
measurable amount of HbF. These findings suggest that intermittent
ketosis might also explain some increases of HbF in type 1 diabetes and
pregnancy.
| |
INTRODUCTION |
IN BOTH STARVATION and poorly controlled
type 1 diabetes, low insulin levels result in augmented lipolysis and
ketogenesis. Ketones, administered in pharmacologic doses, can
reactivate the production of fetal hemoglobin (HbF;
 2 2) in adult patients.1,2 During early childhood, the level of HbF normally decreases to less
than 1% of the total Hb. However, increased levels of HbF are found in
adult life not only in several inherited, eg, -thalassemia, but also
in acquired disorders, eg, type 1 diabetes,3,4 and in
pregnancy.5 It is unknown whether ketones, which are
generated from unrestrained lipolysis in the hypoinsulinemic state,
could account for a clinically measurable increase in HbF. Therefore, we investigated HbF in young women with anorexia nervosa or bulimia, using both of these as models of intermittent starvation
ketosis.6,7
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MATERIALS AND METHODS |
Subjects.
Thirty-one women suffering from anorexia nervosa or bulimia and 42 female control subjects matched for age were studied
(Table 1). The patients with anorexia
nervosa (n = 10) or bulimia (n = 21) were investigated on the day of
hospitalization, before they were started on an inpatient program at
the Medical University of Lübeck. Diagnoses were made according
to DSM-III-R criteria.8 Control subjects were chosen from
among students attending a school of economics. Age was 25 ± 1 years (mean ± SEM) in both groups (range, 18 to 34 years in the
patients and 15 to 39 years in the control subjects). None of the study
subjects had a history of diabetes mellitus, acute blood loss within
the last 8 weeks, present pregnancy, cancer, or hematologic disease, as
these are conditions that might affect the production of
HbF.9 None of the study subjects reported past or present
alcohol or drug abuse. The control subjects had no history or current
symptoms of eating disorders. Before inclusion in the study, every
person signed a consent form which had been approved by the local
ethics commission (for patients <18 years of age, her parents would
sign).
Analytical methods.
Blood samples were drawn after a 12-hour overnight fast for
measurements of -hydroxybutyrate, HbF, and other metabolic and hematologic parameters. Serum -hydroxybutyrate was measured with a
photometric method using a digital photometer 6114 S (Eppendorf Netheler Hinz, Hamburg, Germany). The -hydroxybutyrate
intra-assay coefficient of variation (CV) was 2.1%, and the
corresponding interassay CV was 2.2% (Sigma Diagnostics, Deisenhofen,
Germany). The reference interval for fasting values was 20 to 410 µmol/L in our laboratory.
HbF was measured with the high pressure liquid chromatography (HPLC)
method according to Jeppsson et al.10 The chromatographic system consisted of a 4000 autosampler, an L-6200 intelligent pump, an
L-5025 column heater, and an L-4250 UV-VIS detector (all from E. Merck,
Darmstadt, Germany). System controlling and calculations were performed
with the D-7000-HPLC-Manager software (Merck). Kation-exchange
chromatography was performed with a Mono-S-HR 5/5 column (Kabi
Pharmacia Diagnostics, Uppsala, Sweden). The HbF intra-assay CV was
0.1%, and the corresponding interassay CV was 1.8% at an HbF level of
2.1%. Because HbF continues to decrease throughout adult
life9 and study subjects were in part adolescents, the
reference interval for adults was not valid for our study population.
Therefore, we defined an elevated HbF fraction as a value above the
95% percentile of the control group (HbF >0.87%). HbA1c
was also measured with this HPLC method (normal range, 4.2% to 6.8%).
Other reference intervals for our laboratory were essentially equal to
those listed by Laposata.11
Statistical analysis.
The results are means ± SEM. A sample size of 30 and 40 subjects in
the experimental and control group, respectively, can detect a
difference of 20% in proportion of subjects with elevated HbF with a
power of 0.80 ( = 0.05). To test differences of variables between
the groups, the Mann-Whitney U test was applied. Differences between
the number of subjects with an elevated HbF were analyzed with the
 2 test.
For coping with confounder variables, we applied two strategies: (1)
matching in the design phase and (2) statistical adjustment in the
analysis phase. Because more than one variable was found different in
the two groups, we used a multivariate analysis for adjustment.
Constructing and validating a multivariate model of the effects of
covariates on outcome represents an attempt to remove much or most of
the effects of covariates from the observed results.12 To
avoid possible violations of distribution assumptions (normality,
homoscedasticity),13 we did not apply linear but rather
applied dichotomous logistic regression analysis to our data.14 Because HbF continues to decrease
throughout adult life, age was included in the logistic regression
model irrespective of statistical significance. The variable
-hydroxybutyrate and additional covariates (hematologic and
metabolic abnormalities) were conditionally selected in a forward
stepwise procedure (inclusion criteria, P < .05).
Calculations were performed using the SPSS statistics program Version
6.0 (SPSS Inc, Chicago, IL).
| |
RESULTS |
The clinical characteristics of the two groups were not different as
far as age, sex, HbA1c, Hb, mean corpuscular volume, reticulocyte count, platelet count, iron, ferritin, bilirubin, aspartate aminotransferase, lactate dehydrogenase, haptoglobin, and
potassium were concerned (Table 1). However, -hydroxybutyrate levels
were higher in the patients than in the controls (460 ± 90 v 110 ± 20 µmol/L, P < .0001;
Fig 1A). In addition, we observed distinct
differences in the possible interfering variables body mass index,
leukocyte count, transferrin, hematocrit, and erythrocyte count (Table
1). Subjects with an elevated HbF fraction (>0.87%) were observed
four times as often in patients than in controls (29 v 7%,
P = .0126; Fig 1B).
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| Fig 1.
-Hydroxybutyrate levels (P < .0001; A) and
proportions of subjects with elevated HbF fraction (>0.87%)
(P = .0126; B) in female control subjects and in women
suffering from anorexia nervosa or bulimia.
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|
HbF and -hydroxybutyrate levels were associated, as shown in the
original data plot (Fig 2). We applied
multivariate logistic regression models to assess which variables might
explain the differences in HbF between patients and controls
(Tables 2 and 3). Logistic regression of elevated HbF
(Table 2) showed a significant age-adjusted effect of
-hydroxybutyrate (P = .005). There was no significant effect
of other possible interfering covariates: body mass index, Hb,
hematocrit, erythrocytes, reticulocytes, leukocytes, platelets, iron,
ferritin, transferrin, bilirubin, aspartate aminotransferase, lactate
dehydrogenase, haptoglobin, potassium, and group (patients/controls).
The latter variable "group (patients/controls)" summarizes the
effects of variables that could have accounted for differences in HbF
between patients and controls but have not explicitly been defined in
the present study. The effect of this group-variable was
nonsignificant, making a major effect of unidentified variables
unlikely. To confirm the effect of -hydroxybutyrate, we removed
-hydroxybutyrate from an alternate regression model (Table 3). In
this model, only the effect of the group-variable (patients/controls)
became significant (P = .02). Thus, from among the variables
investigated in this study, -hydroxybutyrate exclusively
explained the differences in HbF elevations between patients and
controls.
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| Fig 2.
Scatterplot of HbF and -hydroxybutyrate levels in
female control subjects ( ) and in women suffering from anorexia
nervosa ( ) or bulimia ( ) .
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| |
DISCUSSION |
Our data show that a modest level of ketonemia, generated from
unrestrained lipolysis, is related to an increased synthesis of HbF.
Specifically, we found a strong association between -hydroxybutyrate and elevated HbF in young women with starvation ketosis. In several other experimental and clinical conditions, ketones have been shown to
induce the HbF production. The discovery that butyrate is an inducer of
HbF arose from the observation that the switch from HbF
(22) to Hb A
(22) is delayed in infants of diabetic mothers. In these newborns, elevated plasma concentrations of a labile
analogue of butyric acid, -amino-n-butyric acid, were reported.15 Second, in vivo and in vitro experiments have
confirmed that butyrates are potent stimuli of -globin mRNA and
protein production.16,17 Third, a clinical pilot trial in
patients with -hemoglobinopathies showed that butyrate can increase
fetal-globin production within weeks to levels that can ameliorate
-globin disorders.1 Finally, a case has been reported
with an induction of HbF in the presence of increased 3-hydroxybutyric
acid associated with -ketothiolase deficiency.18 This
natural in vivo experiment indicated that the induction of HbF requires
a sustained presence of high butyrate concentrations. In addition to
these various conditions, our findings clearly showed that in
starvation ketosis -hydroxybutyrate was generated in sufficient
large concentrations to stimulate a clinically measurable HbF
production.
The mechanism by which butyrate achieves HbF induction is unknown.
Butyrate stimulates a specific embryonic  -globin gene in adult
chickens through 5 flanking sequences19 and
selectively stimulates the -gene in fetal sheep, cultured human
erythroid cells, and adult nonhuman primates.16,17,20
Butyrate may act through sequences near the transcriptional start site
to stimulate the activity of the human -globin gene
promoter.21 Because butyrate is known to inhibit the enzyme
histone deacetylase22 and to increase the acetylation of
histones in other systems, Perrine et al postulated that
this activity could be involved in the action of butyrate on -globin
expression.21 In the biochemical pathway of butyrate
catabolism, butyrate enters mitochondria, where it undergoes
-oxidation to form acetate in a molar ratio of 2:1. Both butyrate
and acetate, as well as chemical derivates of these compounds, can
induce HbF.23,24 We do not know yet whether each of these
compounds acts directly to induce HbF or whether they are converted to
one or more metabolic intermediates that subsequently exert their
effect. Like many conditions resulting in increased HbF in adult life,
ketonemia appears to involve an increased erythropoietic drive that
results in a higher proportion of erythroid progenitor cells activating
their inherent ability to synthesize low amounts of HbF.
In addition to ketones, we have considered other possible interfering
variables in our calculations that are known to occur in eating
disorders, ie, hematologic and metabolic abnormalities. Hematologic
abnormalities are well recognized in anorexia, and several classes of
pancytopenia due to bone marrow hypoplasia and, more rarely, to marrow
cell necrosis have been reported.25,26 These changes have
been shown to be reversible after body weight was restored. In fact, we
found lower leukocyte counts in the anorexia and bulimia patients.
However, no relationship could otherwise be established in our data
between blood cell counts, total Hb, the parameters of the iron
metabolism, hemolysis parameters, and HbF, making an interfering effect
of hematologic abnormalities unlikely. Metabolic abnormalities included
weight loss, hypokalemia, and elevated liver enzymes.27
Similarly, no relationship could have been established between body
mass index, aspartate aminotransferase, serum potassium, and HbF. Thus,
we consider it to be improbable that confounding effects, especially of
hematologic and metabolic abnormalities, could have been the basis of
the observed relationship between -hydroxybutyrate and HbF.
Our findings are consistent with those of previous studies on type 1 diabetes and pregnancy. In type 1 diabetes, ketosis may occur as a
result of poor metabolic control. It is often caused by insufficient
insulin intake but may result from physical (eg, infection) or
emotional stress, despite continued insulin therapy. As a result of
hypoinsulinemia, lipolysis is augmented, and acetoacetic and
-hydroxybutyric acids are produced. Our results suggest intermittent ketosis as a factor that could account for HbF elevations,
as seen in type 1 diabetes.4,28 In pregnancy, ketosis tends
to develop in the presence of "accelerated
starvation."29 The hormonal and substrate milieu after
an overnight 12-hour fast in pregnancy is comparable to that observed
after a 36-hour fast in the nongravid state, hence the term
"accelerated starvation." Similarly, our results suggest ketosis
as a factor that could account for maternal HbF production
as seen in pregnancies.5 To define the possible causes of
HbF elevations in pregnancy is relevant because of the differential
diagnosis of transplacental hemorrhage30 and because
gestational ketonemia imposes a risk on fetal
development.31
In conclusion, we have been able to show that -hydroxybutyrate
generated in starvation is associated with increased levels of HbF.
Thus, unrestrained lipolysis can produce -hydroxybutyrate in
sufficient quantities to induce a clinically measurable amount of HbF.
Our findings suggest that, also in type 1 diabetes and pregnancy,
intermittent ketosis might stimulate HbF production.
| |
FOOTNOTES |
Submitted June 10, 1997;
accepted September 9, 1997.
Address reprint requests to Achim Peters, MD, Medical Clinic 1, Medical
University Lübeck, Ratzeburger Allee 160, D-23538 Lübeck,
Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are indebted to W. Jelkmann, PhD (Institute for Physiology
Medical University, Lübeck, Germany), K. Lorentz, MD
(Institute for Clinical Chemistry, Medical University, Lübeck, Germany), and C. Peters, MD (Clinic for Anaesthesiology, University of
Kiel, Kiel, Germany) for their valuable suggestions.
| |
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Abstract
Introduction
Abstract