O

Fu

Ba

b

c

a

ARAA

KDP1

B

PIP1

G

0d

Rev Esp Med Nucl. 2011;30(1):14–18

riginal Article

DG uptake in brown adipose tissue—A brief report on brown fat with FDGptake mechanisms and quantitative analysis using dual-time-point FDG PET/CT

. Esen Akkasa,∗, D. Gökaslanb, L. Gunerc, N. Ilgin Karabacakc

Ankara Oncology Research and Training Hospital, Department of Nuclear Medicine, Ankara, TurkeyAnkara Yuksek Ihtisas Research and Training Hospital, Department of Nuclear Medicine, Ankara, TurkeyGazi University Medical Faculty, Department of Nuclear Medicine, Ankara, Turkey

r t i c l e i n f o

rticle history:eceived 26 March 2010ccepted 26 May 2010vailable online 10 August 2010

eywords:ual time point imagingET/CT8F-FDGrown fat

a b s t r a c t

Aims: Brown adipose tissue (BAT) is a potential source of false-positive findings on [18F] FDG PET. In thisreport, we have discussed the 18F-FDG uptake mechanisms in BAT and have aimed to determine if dualtime point PET imaging helps to differentiate BAT from malignant lesions.Methods: Patients with dual-time-point PET/CT scans were reviewed retrospectively and 31 cases (11males, 20 females, age: 28.6±9.7) having hypermetabolic BAT were included for this study. 18F-FDGuptake in BAT was quantitatively analyzed by maximum standardized uptake values (SUVmax), andaverage percent change in SUVmax of BAT between early and delayed images was calculated.Results: Compared to the initial scans, 18F-FDG uptakes in BAT in delayed images were higher in 26 of thepatients, and lower in one patient. In terms of body regions, 18F-FDG uptake increased in 80.6%, remainedunchanged in 5.5% and decreased in 13.9% of the body regions. Mean percent change in SUVmax, includingall BAT regions, was 19.8±19.1% while the mean percent increase was calculated as 69±25% in regionswhere progressive accumulation was observed. The increase in SUVmax correlated with the time intervalbetween the two scans.Conclusion: Physiologic 18F-FDG uptake in BAT increases over time and may mimic the behavior ofmalignant lesions on dual time point PET imaging. Without the exact anatomic definition of the CT scan,false positive interpretation of PET data may be possible in cases with atypical BAT.

© 2010 Elsevier Espana, S.L. and SEMNIM. All rights reserved.

Captación de FDG en tejido adiposo marrón – Breve informe sobre grasa marróncon mecanismos de captación con FDG y análisis cuantitativo utilizando PET/TACcon FDG en dos fases

alabras claves:mágenes en dos fasesET/TAC8F-FDGrasa marrón

r e s u m e n

Objetivos: El tejido adiposo marrón (TAM) es un fuerte potencial de falsos-positivos en el 18F-FDG PET.En este informe, comentamos los mecanismos de captación del 18F-FDG en TAM e intentamos averiguarsi la adquisición de imágenes con el PET en dos fases ayuda a diferenciar el TAM de las lesiones malignas.Métodos: Los pacientes sometidos a estudios PET-TAC en dos fases fueron estudiados retrospectivamentey se incluyeron 31 casos (11 hombres, 20 mujeres, edad: 28,6±9,7) con TAM hipermetabólico en el estudio.La captación de 18F-FDG en el TAM fue analizada cuantitativamente, utilizando los valores estandarizadosde captación máxima (SUVmax), y se calculó el cambio medio porcentual en el SUVmax de TAM entreimágenes tempranas y retardadas.Resultados: Comparadas con los estudios iniciales, las captaciones de 18F-FDG en TAM en las imágenesretardadas fueron más altas en 26 de los pacientes y más baja en uno. En cuanto a las regiones del cuerpo,la captación de 18F-FDG aumentó 80,6 %, no cambió en 5,5 % y disminuyó en el 13,9% de las regionesdel cuerpo. El cambio medio porcentual en SUVmax, incluyendo todas las regiones del TAM, fue de 19,8

%±19,1, mientras que el aumento medio porcentual calculado fue de 69±25% en las regiones en que seobservó acumulación progresiva. El aumento en SUVmax correlacionó con el intervalo de tiempo entrelas dos fases.Conclusión: La captación fisiológica de 18F-FDG en el TAM aumenta con el tiempo y puede simular elcomportamiento de lesiones malignas en el PET en dos fases de tiempo. Sin tener la definición anatómicaexacta del estudio TAC, puede haber una interpretación de falso-positivo de los datos del PET en casoscon un TAM atípica.

∗ Corresponding author.E-mail address: burcuesen@yahoo.com (B. Esen Akkas).

212-6982/$ – see front matter © 2010 Elsevier Espana, S.L. and SEMNIM. All rights reseroi:10.1016/j.remn.2010.05.006

© 2010 Elsevier Espana, S.L. and SEMNIM. Todos los derechos reservados.

Introduction

18F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) has within recent years become

ved.

p Med

tmuletwPsbppPiracmimu[t

sbbnit

M

owltrroA1a

twidoPPtmpPHptwoeir

imaging in SUVmax including all BAT regions was calculated as19.8±19.1% and this increase was found to be significant in sta-tistical analysis (p: 0.0001). The mean percent change in [18F] FDGuptake in BAT regions where progressive accumulation of FDG isobserved was calculated as 69±25%. Besides, a linear regression

B. Esen Akkas et al / Rev Es

he most important nuclear medicine imaging modality in theanagement of various cancer types. 18F-fluorodeoxyglucose

ptake in brown fat can be a common source of potentially mis-eading false-positive PET imaging in the thorax, in the neck andspecially in the abdomen.1–3 PET/CT offers the unique opportunityo correlate PET findings with CT anatomy and precise localizationith fusion PET/CT is important in preventing misinterpretation of

ET data as malignancy.4 Hypermetabolic BAT is typically bilateral,ymmetric, mostly seen in the cervical and supraclavicular regions,eing more common in female patients and in the pediatricopulation, especially in the winter time; its identification is oftenossible even at the first glance.1,3 However, the routine use ofET/CT fusion imaging in a large oncology practice has shown thatncreased uptake of [18F] FDG into BAT in the neck and shoulderegion, axillae, mediastinum, perinephric regions may be seen,nd in some cases the uptake may be asymmetric and withoutorresponding CT slices [18F] FDG uptake can be misinterpreted asalignant/metastatic disease especially in the mediastinum and

n the abdomen.4,5 Even in some problematic cases, lymph nodesay exist in the mediastinal fat tissue and with the [18F] FDG

ptake in BAT, deciding whether the lymph node has pathological18F] FDG uptake may not be possible without some interventionso decrease the [18F] FDG uptake in BAT.

In recent reports, dual-time point [18F] FDG imaging has beenhown to be useful in the differentiation between malignant andenign lesions, since the [18F] FDG uptake in malignant lesions haseen shown to increase over time.6–8 The purpose of this prelimi-ary report was to evaluate the contribution of dual time point PET

maging to differential diagnosis in patients with BAT uptake, ando discuss the [18F] FDG uptake mechanisms in BAT.

ethods

All PET/CT scans (n: 1032) from January to October 2008btained in our institution were retrospectively reviewed. Patientsho were scanned twice for dual time point imaging (78±20 min

ate imaging) for the accurate interpretation of FDG uptakes relatedo their primary pathologies in neck and mediastinum regions weree-evaluated. Among them, cases (n: 31; 11 males, 20 females, ageange: 28.6±9.7) with [18F] FDG uptake in the mediastinum andr cervical regions localized to fat were included for this study.dditional 2 h delay images were then compared with the initialh images for the changes in [18F] FDG uptakes localized to browndipose tissue in 36 body regions.

Patients were fasted for at least 6 h prior to intravenous injec-ion of 5.3 MBq/kg (144 �Ci/kg) [18F] FDG. The blood glucose levelsere less than 160 mg/ dl in all patients at the time of the FDG

njection. Patients were kept lying in supine position in a quiet andim lighted room at normal temperature. All patients were imagedn an integrated PET/CT scanner (Discovery ST-8, GE Healthcare).ET scans were acquired in the 2D mode for 3 min per bed position.ET images were reconstructed both with and without attenua-ion correction. Unenhanced CT images were acquired in the helical

ode (speed, 13.5 mm/rotation) from the base of the skull to theelvis using 3.75-mm slice thickness, 140 kVp and 120 mA. TheET and CT images were reviewed on a workstation (Xeleris, GEealthcare) in all standard planes along with maximum-intensity-rojection images and were analyzed visually and quantitatively bywo reviewers experienced in interpreting PET/CT scans. Findings

ere recorded by consensus. All cases in which patient breathing

r motion resulted in misregistration of increased FDG uptake werexcluded. In addition, a pixel region of interest (ROI) was outlinedn the regions of increased FDG uptake, and after correction foradioactive decay.

Nucl. 2011;30(1):14–18 15

In this retrospective study, patients who were scanned by dualtime point imaging for the better differentiation of the findingsthat may be related to their primary pathologies seen on the PETdata and having FDG uptake in their BAT were selected. As patientswere not exposed to additional radiation dose for this study, ethicalcommittee approval was not needed.

Statistical analysis

Data are expressed as mean and standard deviations. The sta-tistical analysis was performed using SPSS 13.0 programmer. Thedifferences between early and delayed SUVmax were analyzed withWilcoxon Rank Analyses test. The significance of percent changeswas analyzed with an independent samples t test; p values <0.05were considered significant. Linear regression analysis was per-formed to evaluate the correlation between the time interval ofthe PET scans and the percent changes in SUVs.

Results

Patients’ characteristics and brown fat localizations as well asthe FDG uptakes in terms of SUVmax are demonstrated in Table 1.Compared to the initial scans, [18F] FDG uptakes in BAT in delayedimages were higher in 26 of the patients, lower in one patient, andremained unchanged in one patient; both increase and decrease indifferent BAT localizations were observed in 3 patients. In termsof body regions, [18F] FDG uptake increased in 29 regions (80.5%)remained unchanged in 2 regions (5.6%) and decreased in 5 bodyregions (13.9%; Figs. 1 and 2).

At the initial 1 h images, the SUVmax for BAT ranged from3.66 to 17.3, where the mean value was 7.13±3.08. The SUVmaxin delay images ranged from 4.90 to 17.60 and the mean valuewas 8.44±3.59. The overall percent increase by dual-time-point

Fig. 1. Transaxial PET (A), CT (B), fused PET/CT (C) and maximum intensity projec-tion (MIP) (D) images of the initial PET/CT study of patient number 9. IncreasedFDG uptake is observed in bilateral cervical and supraclavicular regions on the MIPimage. Corresponding CT scan shows no abnormal lymph node or muscle uptake insupraclavicular region and 18F-FDG uptake appears to fuse predominantly into fattissue. SUVmax for brown fat tissue was 4.5 on left supraclavicular region.

16 B. Esen Akkas et al / Rev Esp Med Nucl. 2011;30(1):14–18

Table 1Patients characteristics and SUV values with percent changes derived from the early and late images defined for brown fat regions

Case no. Age Diagnosis Gender Region SUVmax early SUVmax late Increase (%) Time interval (min)

1 23 Lymphoma F Supraclavicular 17.3 17.6 1.7 74Mediastinal 5.2 6.8 30

2 27 Lymphoma F Supraclavicular 6.4 8.1 26 1163 40 Breast cancer F Cervical 14 17.4 24 654 23 Seminoma M Cervical 5.5 6.1 10 885 37 Malignant melanoma F Mediastinal 8.1 10.2 25 1026 19 Lymphoma F Mediastinal 5.5 5.9 7 987 40 Breast cancer F Mediastinal 4.1 5.9 43 1118 45 Colorectal carcinoma F Supraclavicular 10.2 12.8 25 759 29 Breast cancer F Supraclavicular 4.5 5.7 26 90

10 14 Osteosarcoma M Supraclavicular 8.70 8.03 −8 60Paravertebral 4.63 4.90 6

11 15 Ewing sarcoma M Mediastinal 5.29 5.13 −3 80Mediastinal 4.31 5.65 31

13 22 Lymphoma F Paravertebral 5.86 5.87 0 6514 36 Lung cancer M Paravertebral 7.9 10.1 28 68

Cervical 4.96 4.96 015 27 Nasopharynx cancer F Cervical 5.54 6.63 20 5216 24 Lymphoma F Supraclavicular 5.4 9.1 68 7517 26 Lymphoma F Cervical 12.7 15.3 20 12018 40 Breast cancer F Cervical 5 6.9 38 6019 23 Lymphoma M Cervical 8.4 11 30 9020 38 Breast cancer F Axillae 7.3 9.5 30 11021 44 Colorectal carcinoma F Supraclavicular 6.3 9.1 44 7822 30 Ovarian cancer F Supraclavicular 8.56 7.48 −13 9023 14 Malignant mesenchymal tumor M Axillae 5.38 5.02 −7 60

Supraclavicular 9.99 12.28 2324 15 Ewing sarcoma M Mediastinal 5.83 7.48 28 8025 18 Nasopharynx cancer F Supraclavicular 3.66 5.35 46 7826 40 Breast cancer F Supraclavicular 5.73 7.76 35 7527 36 Malignant melanoma F Supraclavicular 5.68 8.92 57 10028 35 Lymphoma F Supraclavicular 7.3 9.3 27 100

viculaebralvicula

aissi

Fpptf

29 39 Breast cancer M Cervical30 14 Ostesarcoma M Supracla

Paravert31 26 Seminoma M Supracla

nalysis demonstrated a positive correlation between the percent

ncrease in FDG uptake and the time interval of early and delay PETcans (p: 0.005). In other words, we observed that causing progres-ive accumulation, [18F] FDG uptake tended to increase over timen BAT (Fig. 3).

ig. 2. Transaxial PET (A), CT (B), fused PET/CT (C) and MIP (D) images of dual timeoint PET/CT study obtained 90 min after the initial scan of patient number 9. Com-ared to the initial scan (Fig. 1), increase in FDG uptake is seen visually in brown fatissue and SUVmax is found to be elevated. (SUVmax of left supraclavicular brownat tissue is 5.7).

6.5 7.8 20 106r 5.47 5.85 7 75

6.83 5.36 −22r 12.1 15.1 24 60

Discussion

In previous reports, hypermetabolic BAT was localized to the

paratracheal, paraesophageal, prevascular and pericardial regionsas well as associated extramediastinal hypermetabolic brown fat inthe cervical region paravertebral region and axillary regions.1–4,9

The appearance of [18F] FDG uptake in mediastinal and in the

Fig. 3. Graphic demonstrates the correlation between time interval and percentchanges of FDG uptake in BAT regions. A linear regression analysis showed signifi-cant increase in SUVmax values over time with p: 0.005.

B. Esen Akkas et al / Rev Esp Med

Fig. 4. PET/CT study of patient number 20. She was referred to PET/CT imagingfor restaging of recurrent breast cancer in her previously operated left breast. In theaxial PET slices (A) of the PET/CT study, hypermetabolic foci suggestive of metastaticdisease is seen in the mediastinum and in bilateral axillae prominent in the left side.In the corresponding CT slices (B), pathological lymph nodes in the mediastinum areclearly seen but no lymph nodes in axillary regions can be demonstrated. In fusedPET/CT image (C), the hypermetabolic foci in bilateral axillae suit to fat tissue. Inste

apiialFnuiawcthlcIttiltabt

BiowBtp(pn

uagWaB

the PET scans. Today, fusion PET/CT provides the unique advantage

uch a case with recurrent breast cancer, the unilateral axilla had a high potentialo have metastatic lymph nodes and the false positive interpretation of the axillae (specially left axilla) could be possible if PET was be considered alone.

bdominal brown fat may be focal, curvilinear or spherical. Someatients may have an isolated single focus of hypermetabolic BAT

n the mediastinum/abdomen or even in subcutaneous tissue—thats, without hypermetabolic BAT elsewhere in the body.4,10 Thesereas are most likely to be concerned for the accurate staging ofymphoma and with PET alone, focal increased uptake of [18F]DG in BAT may be misinterpreted as primary esophageal malig-ancy, primary lymphoma or nodal metastases, leading to tumorpstaging or unnecessary medical or surgical interventions. The

ncreased FDG uptake in BAT in the neck region has been reporteds a cause of misinterpretation in 2.3–4% of patients.1,11 Especiallyhen located in the mediastinum and in the upper abdomen, BAT

an be a potential pitfall for misinterpretation.1,4,5,10,12 Althoughhe development and widespread use of integrated PET/CT systemsas lead the PET clinicians to overcome this potential pitfall to a

arge extent, in some problematic cases the FDG uptake in BAT stillontinues to complicate the evaluation of the PET data (Fig. 4).10,12

n this regard, in order to differentiate benign versus malign lesions,he authors report the benefit of using dual-time-point imaging dueo the increased [18F] FDG uptake in malignant tumors in delayedmages. On the other hand, physiologic [18F] FDG uptake (tongue,arynx, muscles) and [18F] FDG uptake of inflammatory lesions tendo decline over time.7,8 Thus, in the view of these dual imagingpproaches, brown fat has naturally been expected to show similarehavior to other benign and physiologic conditions when the dualime point imaging principles are applied.

In this report, we observed that physiologic 18-F FDG uptake inAT has increased in the delayed images in the majority of local-

zations (80.5%) contrary to the expected decrease as observed inther benign conditions that are previously reported. Althoughe observed reduced or unchanged SUVs in a small number ofAT localizations, no specific differences were present amonghe patients characteristics (including age, sex, primary diagnosis,atient blood glucose level) and/or PET/CT acquisition parametersincluding scan time, delay between the two scans and room tem-erature). We considered that these observations above could beormal variants.

When we prearranged the study, we hypothesized that FDGptake in BAT would show a decrease as expected in other benign

nd physiological conditions. But interestingly, we observed pro-ressive accumulation of FDG, which is correlated by time in BAT.e have no definite explanations but there may be several mech-

nisms and reasons for this interesting metabolic phenomenon.rown adipose tissue (BAT) or brown fat is one of the two types of

Nucl. 2011;30(1):14–18 17

adipose tissue (the other being white adipose tissue) that is presentin many newborn or hibernating mammals. Its primary purpose isto generate body heat. In contrast with white adipocytes (fat cells),which contain a single, large fat vacuole, brown adipocytes containseveral smaller vacuoles and a much higher number of mitochon-dria. Brown fat also contains more capillaries since it has a greaterneed for oxygen than most tissues. BAT differs from regular adiposetissue by its high content of mitochondria and plays an importantrole in the regulation of body temperature in hibernating mam-mals and in newborns.9 Brown fat can use glucose as a substrateespecially when activated. Since the primary function of BAT is togenerate body temperature, the acceleration of glucose utilizationwith cold exposure is an expected result, as mentioned in previousreports.13–15 The mitochondria in an eukaryotic cell utilize fuels toproduce energy (in the form of ATP). This process involves storingenergy as a proton gradient, also known as the proton motive force(PMF), across the mitochondrial inner membrane. This energy isused to synthesize ATP when the protons flow across the membrane(down their concentration gradient) through the ATP synthetaseenzyme. This model is known as the chemiosmotic hypothesis.

In endothermic animals, body heat is maintained by signalingthe mitochondria to allow protons to run back along the gradientwithout producing ATP. This can occur since an alternative returnroute for the protons exists through an uncoupling protein in theinner membrane. This protein, known as thermogenin, or uncou-pling protein 1, facilitates the return of the protons after they havebeen actively pumped out of the mitochondria by the electrontransport chain. This alternative route for protons uncouples oxida-tive phosphorylation and the energy in the PMF is released as heat.To some degree, all cells of endotherms give off heat, especiallywhen body temperature is below a regulatory threshold; however,brown adipose tissue is highly specialized for this non-shiveringthermo genesis. Firstly, each cell has a higher number of mitochon-dria compared to more typical cells. Secondly, these mitochondriahave a higher than normal concentration of thermogenin in theinner membrane. These seem to be the possible mechanisms andreasons for high and increasing FDG uptake over time in BAT.

In this retrospective study, only patients who were scannedwith dual time point PET imaging for the accurate interpretationof FDG uptakes in neck and mediastinum were selected and noneof them were scanned twice just for the observation of FDG uptakesin brown fat localizations. Among them, we observed mediastinalBAT in 7 patients, cervical and supraclavicular BAT in 22 patientsbut no abdominal BAT is observed. In this study none of the caseswere misinterpreted as malignancy with the typical appearance ofFDG uptake and with the exact anatomic definition by CT. Althoughwe have not designed this study to suggest the use of dual timepoint PET/CT imaging for the differential diagnosis of FDG uptakein brown fat, we found progressive accumulation of FDG uptake inbrown adipose tissue by time, which is contrary to what is expectedin other benign/physiological conditions worth reporting.

Conclusion

In this brief report, although symmetrical and easy to recognizein the majority of cases, we observed that physiologic FDG uptakein brown adipose tissue increases over time on dual time pointPET imaging and mimics the behavior of malignant lesions. Addi-tionally, this increase is correlated with the time interval between

to give anatomical information and CT component contributes tothe specificity of PET/CT imaging. But it must be kept in mind thatby dual time point imaging, the observed uptake phenomenon ofFDG in BAT may indeed lead to misdiagnosis in cases where theappearance of BAT is atypical.

1 sp Med

C

R

8 B. Esen Akkas et al / Rev E

onflict of interest

The authors affirm that they have no conflicts of interest.

eferences

1. Cohade C, Osman M, Pannu HK, Wahl RL. Uptake in supraclavicula area fat(“USA-Fat”): description on 18F-FDG PET. J Nucl Med. 2003;44:170–6.

2. Shreve PD, Anzai Y, Wahl RL. Pitfalls in oncologic diagnosis with FDG PET imag-ing: physiologic and benign variants. Radiographics. 1999;19:61–77.

3. Hany TF, Gharehpapagh E, Kamel EM, Buck A, Himms-Hagen J, von SchulthessGK. Brown adipose tissue: a factor to consider in symmetrical tracer uptake inthe neck and upper chest region. Eur J Nucl Med Mol Imaging. 2002;29:1393–8.

4. Truong MT, Erasmus JJ, Munden RF, Marom EM, Sabloff BS, Gladish GW, et al.Focal FDG uptake in mediastinal brown fat mimicking malignancy: a potentialpitfall resolved on PET/CT. Am J Roentgenol. 2004;183:1127–32.

5. Hyun IY, Kim SG. FDG uptake in parahepatic brown fat mimics peritoneal carci-

nomatosis in a malignant ovarian germ cell tumor: resolution with temperaturecontrol. Clin Nucl Med. 2008;33:799–801.

6. Matthies A, Hickeson M, Cuchiara A, Alavi A. Dual time point 18F-FDG PET forthe evaluation of pulmonary nodules. J Nucl Med. 2002;43:871–5.

7. Hustinx R, Smith R, Benard F, Rosenthal DI, Machtay M, Farber LA, et al. Dualtime point fluorine-18 fluorodeoxyglucose positron emission tomography: a

Nucl. 2011;30(1):14–18

potential method to differentiate malignancy from inflammation and normaltissue in the head and neck. Eur J Nucl Med. 1999;26:1345–8.

8. Zhuang H, Pourdehnad M, Lambright ES, Yamamoto AJ, Lanuti M, Li P, et al. Dualtime point 18F-FDG PET imaging for differentiating malignant from inflamma-tory processes. J Nucl Med. 2001;42:1412–7.

9. Cannon B, Nedergaard J. Brown adipose tissue: function and physiological sig-nificance. Physiol Rev. 2004;84:277–359.

10. Clarke JR, Brglevska S, Lau EW, Ramdave S, Hicks RJ. Atypical brown fat dis-tribution in young males demonstrated on PET/CT. Clin Nucl Med. 2007;32:679–82.

11. Yeung HW, Grewal RK, Gonen M, Schöder H, Larson SM. Patterns of (18)F-FDGuptake in adipose tissue and muscle: a potential source of false-positives forPET. J Nucl Med. 2003;44:1789–96.

12. Reddy MP, Ramaswamy MR. FDG uptake in brown adipose tissue mimickingan adrenal metastasis: source of false-positive interpretation. Clin Nucl Med.2005;30:257–8.

13. Greco-Perotto R, Zaninetti D, Assimacopoulos-Jeannet F, Bobbioni E, Jeanre-naud B. Stimulatory effect of cold adaptation on glucose utilization by brownadipose tissue: relationship with changes in the glucose transporter system. J

Biol Chem. 1987;262:7732–6.

14. Christensen CR, Clark PB, Morton KA. Reversal of hypermetabolic brown adi-pose tissue in F-18 FDG PET imaging. Clin Nucl Med. 2006;31:193–6.

15. López-Soriano FJ, Fernández-López JA, Mampel T, Villarroya F, Iglesias R, Ale-many M. Amino acid and glucose uptake by rat brown adipose tissue. Effect ofcold-exposure and acclimation. Biochem J. 1988;252:843–9.