Yonago Acta medica 1997;40:13–19

Plasma Lactic Acid and Pyruvic Acid Levels in Patients with Chronic
Primary Headaches during the Headache Free Period
Hiroko Okada, Shigeru Araga, Takao Takeshima and Kenji Nakashima
Division of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori
University, Yonago 683, JapanTo study mitochondrial abnormalities in patients with chronic primary headaches, lactic
acid (LA) and pyruvic acid (PA) levels in plasma were measured. Subjects were 14
patients with migraine [age, 38.0 ± 14.7 years (mean ± SD); male/female ratio, 6/8; 3
with aura and 11 without aura] and 17 patients with tension-type headache (TH) (age,44.5 ± 19.5 years; male/female ratio, 8/9; 3, chronic and 14, episodic) during headache
free periods; 12 healthy volunteers [age, 36.3 ± 8.4 years; male/female ratio, 6/6] served
as controls. The plasma LA level was measured with the lactate oxidase-peroxidase
method, and the plasma PA level, with the pyruvate oxidase peroxidase method. The
migraine, TH and control groups showed 9.6 ± 5.0 mg/dL, 7.2 ± 5.8 mg/dL and 3.3 ± 1.9
mg/dL as the mean plasma LA, respectively; and 0.51 ± 0.30 mg/dL, 0.47 ± 0.45 mg/dL
and 0.26 ± 0.20 mg/dL as the mean plasma PA, respectively. The migraine group showedsignificantly higher means for plasma LA and PA than the control group. The meansin the TH group were lower than in the migraine group and higher than in the control
group, without significant differences. These results may support the hypothesis that
migraine patients have functional abnormalities in their mitochondrial energy
metabolism.
Key words: energy metabolism; migraine; mitochondrial dysfunction; tension-type headache

Migraine and tension-type headache (TH) are common forms of chronic primary headaches.

migraine headache, we focused on one which
describes mitochondrial dysfunction(Montagna et al., 1988, 1994; Bresolin et al.,
1991; Sangiorgi et al., 1994; Watanabe et al.,1994). Plasma pyruvic acid (PA) and lactic
acid (LA) levels can reflect anaerobic andaerobic glycolysis, and the energy metabolism
in mitochondria. In this paper, to clarify
whether or not patients with chronic primaryheadaches have mitochondrial abnormalities,
we examined the PA and LA levels in theplasma of patients with migraine and TH during
headache free periods.

Approximately half of the patients visiting
neurology clinics suffer from primary rather
than non-organic headaches. The life prognosis
of primary headache syndrome is essentially
good; however, these headaches annoy patientsand disturb their quality of life. The etiology of
migraine and TH still remains uncertain. Some
compounds are known to be useful in the
management of headaches, but most therapeutic
methods remain for symptomatic treatment.
Studies aiming to reveal the pathophysiology
and mechanism of headache and a search forrelevant therapies should be carried out.
Among the hypotheses on the etiology ofAbbreviations: ANOVA, analysis of variance; 5-HIAA, 5-hydroxyindole acetic acid; IHS, International Headache
Society; LA, lactic acid; MELAS, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes; PA,
pyruvic acid; TH, tension-type headache13

H. Okada et al.

Table 1. Background of patients and healthy controls

Patients with migraine
Patients with TH
Healthy controls* Mean ± SD.
TH, tension-type headache.

No. of subjects

Male/female ratio

Age*

14
17
12

6/8
8/9
6/6

38.0 ± 14.7
44.5 ± 19.5
36.3 ± 8.4

Subjects and Methods

TH were established in accordance with International Headache Society (IHS) criteria(Headache Classification Committee of the
IHS, 1988). Thus, the participants were required to satisfy all of the following qualifications:i) fulfilling the IHS criteria for the diagnosis of
migraine or TH; ii) giving informed consent;iii) free from daily basic medication for at least
1 month; and iv) free from temporary use ofdrugs at least 5 days before blood sampling.

Subjects
Serial headache sufferers visiting the neurology
clinic of Tottori University Hospital and fulfilling the diagnostic criteria were requested toparticipate in this study. Fourteen patients with
migraine and 17 patients with TH gave theirinformed consent after they fully understood thenature and scope of this study. Twelve healthy
persons volunteered for this study as healthy controls. The background of the participants are
summarized in Table 1. The patients’ headachecharacteristics and the accompanying symptoms are summarized in Table 2. No participants received any prophylactic medication
for at least 1 month before blood sampling. All
patients were free from the abortive use ofdrugs at least 5 days before blood sampling.
Diagnostic criteria of migraine headache andBlood sampling
To exclude the influence of probable diurnal
variation and exercise, we obtained 1.0 mL
blood between 09:00 and 11:00 in the sitting
position after more than 15 min rest, by puncture of the cubital vein with a 21 gauge needle
and a 2.5 mL plastic syringe. Blood samples of
0.5 mL were immediately transferred to 1.5 mL
Eppendorf tubes containing 0.5 mL of 0.8 N

Table 2. Patient characteristics of headaches with associated symptoms
Migraine patientWith aura Without aura
No. of patients
Male/female ratio
Duration of headache
Frequency of headache attacks
Duration of an attack(year)(/month)(h)

Associated symptoms(%)
Nausea and/or vomiting
Dizziness
Photophobia and/or phonophobia
Muscle stiffness of neck and shoulder[ ], range.
TH, tension-type headache.
*This disease is chronic TH.

TH patient
Episodic
Chronic

3
1/2
18.7[ 6.0–30]
1.1[ 0.2– 2]
20.0[12.0–24]

11
5/6
18.0
[1.0–40]
4.8[1.0–14]3.0[0.3–24]

14
6/8
5.5
[ 0.5–25]
2.7[ 0.5–10]
41.6[ 2.5–96]

3
2/1
15.0
[ 5.0–20]
20.0[20.0]
*
*

33.3
33.3
33.3
33.3

57.1
42.9
71.4
36.4

20.0
10.0
14.3
55.6

0
33.3
0
100.0

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Plasma LA and PA

cooled perchrolic acid (Wako Pure Chemicals,
Osaka, Japan). The tubes containing the samples were vortexed and kept on ice for few
hours. Deproteinized supernatant plasma by
centrifugation (1200 × g 15 min) was stocked at–20˚C until use for assay.

LA concentration (mg/dL) =(Es/Estd) × standard plasma concentration,
where, Es = absorption of samples and Estd = absorption of the standard plasma. The assay
coefficient variation was 5.0% and the measurement was linear up to 130 mg/dL. All samples
were encoded and assayed in blind.

Estimation of plasma LA levels
Estimation of plasma PA levels

Plasma LA levels were measured with the lactateoxidase-peroxidase method. All chemicals werepurchased as a Determiner LA kit (Kyowa
Medics, Tokyo, Japan). In brief, 6 µL each of patient plasma samples and 40 mg/dL of thestandard plasma were taken into a 1.5 mL
eppendorf tube, and then the N-ethyl-N-(3methylphenyl)-N'-acetylethylenediamine
solution was added and incubated at 37°C in awater bath for 3 min. A solution of peroxidase
was added, and the mixture was incubated at
37°C in a water bath for 4.5 min. The color intensity (l max = 555 nm) was immediately
measured at 660/600 nm by a dual length spectrophotometer, UV 160 (Shimadzu, Tokyo, Japan).
The LA concentration was calculated using thefollowing formula:

The pyruvate oxidase-peroxidase method wasused in measuring plasma PA levels. All
chemicals were purchased as a Determiner PA
kit (Kyowa Medics). All procedures and thecalculation formula were essentially the sameas the LA measurement except for the color intensity (l max=755 nm). All samples wereencoded and assayed in blind.

Statistical analysis
Statistical analysis of data was performed withthe one-way analysis of variance (ANOVA),the Student-Newmann-Kleus post hoc test andsimple regression. P values less than 5% wereconsidered to be significant throughout thisstudy.

Results

Plasma LA levels
The migraine, TH and control groups showed
9.6 ± 5.0 mg/dL, 7.2 ± 5.8 mg/dL and 3.3 ± 1.9
mg/dL as the mean plasma LA, respectively.
All values of patient samples and mean bars areillustrated in Fig. 1. The migraine group showed a significantly higher mean of plasma LA
than the control group (P < 0.01 ANOVA, theStudent-Newmann-Kleus post hoc test): therelation between the existence of aura symptoms and plasma LA levels was not significant.
In the TH group, plasma LA levels varied from1.04 mg/dL to 20.94 mg/dL, with the mean not
significantly different from that in the migraine
or control group. The plasma LA level showedno significant relation to the duration of headache or frequency of headache attacks.

Fig. 1. Plasma lactic acid (LA) levels in groups ofhealthy controls (n = 12), patients with migraine (n =14) and patients with tension-type headache (TH) (n= 17). The horizontal bar in each group represents
the mean value.
, healthy control; , migrainewith aura; , migraine without aura; , chronic TH;, episodic TH.

15

H. Okada et al.

glucose 6-phosphate, which is accomplished by
the enzyme hexokinase and by an additional
enzyme in the liver, glucokinase. ATP is required as a phosphate donor, and reacts as the
Mg-ATP complex. Finally 1 mol of glucose produces 2 mol of PA and 2 mol of ATP following
enzymatic steps. LA dehydrogenase converts
PA to LA. PA enters a tricarboxylic acid cycle,
which is an aerobic process of glycolysis, in the
mitochondria. PA and LA levels in plasma increase in some pathological conditions such as
hard exercise, hepatic and renal dysfunction,
diabetes mellitus and mitochondrial diseases(Cohen and Simpson, 1975). Plasma LA andPA levels at rest can reflect congenital or acquired mitochondrial dysfunction.
Montagna and others (1988) first reported
that during effort, blood lactate rises moresignificantly in patients with migraine than incontrols and suggested impaired mitochondrialenergy metabolism in migraine. They also reported that by using magnetic resonance spectroscopy (MRS) technique (Montagna et al.,1994), brain energy metabolism in migraine
appears abnormal. Watanabe and others (1994)
reported that MRS was performed in a woman
with migraine attacks accompanied by righthemiplegia and responsive lesion of the brain
who showed elevated LA levels in the left lobe.
Our data presented here agree with earlier reports which suggest dysfunction of energy metabolism in migraine.

Fig. 2. Plasma pyruvic acid (PA) levels in groups ofhealthy controls (n = 12), patients with migraine (n =14) and patients with tension-type headache (TH) (n= 17). The horizontal bar in each group represents
the mean value.
, healthy control; , migrainewith aura; , migraine without aura; , chronic TH;, episodic TH.

Plasma PA levels
The migraine, TH and control groups showed 0.51
± 0.30 mg/dL, 0.47 ± 0.45 mg/dL and 0.26 ± 0.20
mg/dL as the mean plasma PA, respectively.
Figure 2 shows all data of plasma PA in headache
patients and controls with mean bars. The mean
PA was significantly higher in the migraine groupthan in the control group (P < 0.05 ANOVA, theStudent-Newmann-Kleus post hoc test).

Correlation between plasma PA andplasma PA
The plasma LA concentration was significantly
correlated with the plasma PA concentration(simple regression: r = 0.489, P < 0.01) (Fig. 3).

Discussion
PA is synthesized in an anaerobic process of
glycolysis from glucose or glycogen, which is
known as the Embden-Myerhof pathway(Harper et al., 1939). Glucose enters into a
glycolytic pathway by phosphorylation into

Fig. 3. Correlation between plasma lactic acid (LA)levels and plasma pyruvic acid (PA) levels in all
subjects (n = 43).

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Plasma LA and PA

Some congenital or hereditary mitochondrial diseases, including chronic progressive
external ophtalmoplegia (Tome and Fardeau,1986), mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS)(Pavlakis et al., 1984; Montagna et al., 1988)
and mitochondrial encephalopathy with ragged
red fibers (Fukuhara et al., 1980) are identified
mutations of mitochondrial DNA (Holt et al.,
1988; Moraes et al., 1989; Goto et al., 1990;
Kobayashi et al., 1990; Shoffner et al., 1990;
Hammans et al., 1993). Patients suffering fromMELAS, who bears a mutation of mitochondrial DNA at 3243 or 3271, frequently havemigraines with severe headaches and vomiting(Hirano et al., 1992). Some members of MELAS
families have only migraine-like headaches, butneither encephalopathy nor myopathy. One
possibility is that migraine is one of the monosymptomatic forms of MELAS. On the basis of
this possibility, there have been some efforts to
identify mitochondrial DNA mutation in
migraine. Bresolin and others (1991) reported a
mitochondrial DNA deletion in one of 6 patients examined who suffered from migrainous
stroke. Klopstock and others (1996) reported
that they found no mitochondrial mutations in
23 patients with migraine. According to theirobservation, it is unlikely that migraine is acongenital mitochondrial disorder. They also
concluded that their data were strongly against
a hypothesis that some cases of migraine may
be monosymptomatic forms of a MELAS syndrome.
If dysfunction of the mitochondria is notcongenital, we should consider the acquired
dysfunction of mitochondria and the possible
relation to earlier observations of migraine
pathogenesis. As we mentioned in the introduction, the etiology of migraine remains unknown. However, some clues and working
hypotheses have been submitted. Three major
hypotheses of migraine are: i) the neural theory,ii) the vascular theory and iii) the trigeminovascular theory. The neural theory claims that
hyperexcitability of the central nervous systemcauses aura symptoms and cortical spreading
depression, with subsequent vascular throbbing
headaches (Leao, 1944). The vascular theory

claims that vasoconstriction causes local
ischemia of the brain (Wolff, 1963), which is
responsible for aura symptoms, such as scintillating scotoma, hemiplegia and/or aphasia. The
following vasodilatation causes throbbing
headaches. Abnormal platelet serotonin metabolism plays a crucial role in the alteration of vascular tone in migraine headaches (Hanington,1978). The released serotonin from one platelet
stimulates other platelets to release serotonin.
The chain reaction of serotonin release causes
high serotonin levels in the blood and vasoconstriction. Afterwards, the serotonin in the bloodis metabolized into 5-hydoxyindole acetic acid
(5-HIAA). 5-HIAA is secreted into the urine
and blood and the platelet serotonin level
decreases, which causes vasodilatation andthrobbing headaches. The trigemino-vasculartheory claims that an unknown neurochemical
noxious trigger stimulates the trigeminal nerveand causes hyperactivity of trigeminal nerves(Moskowitz, 1984). Substance P and otherneurotransmitters are released from smalldiameter trigeminal sensory afferents aroundthe meningeal and dural blood vessels, initiating vasodilatation, plasma extravasation, andrelease of histamine from mast cells and serotonin from platelets. Serotonin causes vasoconstriction and local ischemia of the brain.
The brain metabolism depends mostly on a
mitochondrial energy supply. Ninety-five
percent of the molecular oxygen is metabolized
within the mitochondria by the electron transport system so that the mitochondria are highly
exposed to oxidative stress. Oxygen radicals
created during respiration can induce mitochondrial dysfunction which accelerates the production of a more deleterious species of oxygen.
Such a mal-cycle due to ischemia-mitochondrial
damage might produce a migrainous state. As
mentioned above, the trigemino-vasculartheory of migraine hypothesizes an unknown
noxious stimulus. Metabolic acidosis resulting
from chronic ischemia and increasing LA andPA may be one of the hypothesized trigger
stimuli in the trigemino-vascular theory.
According to the IHS criteria, migraine andTH are different entities. However, some researchers suspect that migraine and TH are17

H. Okada et al.

similar and may have a common etiology (Bakal
and Kaganov, 1977; Cohen, 1978; Featherstone,1985). Actually, many patients with migraine,
whose diagnoses have been established by the
IHS criteria, frequently have TH. Featherstone(1985) stated that quantification of symptoms
and characteristics showed unipolar distribution, and that therefore, migraine and muscle
contraction (tension-type) headache were acontinuum. Some researchers reported thatsome evidence supports this hypothesis fromthe viewpoint of pupillary function evaluatedby video-pupillometry (Takeshima et al., 1987),
plasma norepinephrine response to cold stimulus (Takeshima et al., 1989), cardiovascular reflex response by orthostastic test (Mikamo etal., 1989), platelet function (Rolf et al., 1981),
prevalence of muscle tenderness (Tfelt-Hansen
et al., 1981) and exteroceptive suppression(ES2) of muscle activity (Nakashima and
Takahashi, 1991). In TH patients, LA and PA
levels showed varying degrees of concentration. Some patients showed high levels in boththe LA and PA concentration in the plasma. In
this regard, continuous muscle contraction in
TH patients may elevate the LA and PA levels.
The mean plasma LA and PA levels in the TH
group were between those in the migraine andcontrol groups. Another possibility is that TH
patients have milder mitochondrial abnormalities than migraine patients have.
We observed significantly increased plasma
PA and LA levels in patients with migraine
during headache free periods. These observations support the hypothesis that patients with
migraine have some abnormalities in mitochondrial energy metabolism and mitochondrial
function in an aerobic process of glycolysis.

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