William G. Bradley, Jr., M.D., Ph.D. Effect of Methemoglobin Formation Paul G. Schmidt, Ph.D. on the MR Appearance of Subarachnoid Hemorrhage1 Subarachnoid hemorrhage has a much T HE EARLIEST reports of magnetic resonance (MR) images of in- higher intensity in magnetic resonance tracranial hemorrhage (1, 2) suggested a relatively intense ap- (MR) images with the passage of time. peanance of the hemorrhage relative to the surrounding brain. This Acute subarachnoid hemorrhage is diff i- was attributed to the short Ti relaxation time of the presumably cult to see; within 1 week its appearance paramagnetic, iron-containing hemoglobin. Figure 1 illustrates this has become intensified on Ti-weighted appearance in a patient imaged 1 week after rupture of an aneurysm images. Different concentrations of blood in an anterior communicating artery, with resulting intraparenchy- and lysed red blood cells in cerebrospinal mal hematoma and subarachnoid hemorrhage. The short Ti charac- fluid (CSF) were examined spectroscopi- ten of the injured area is enhanced relative to surrounding brain on cally but did not significantly alter Ti a Ti-weighted spin-echo image. and T2 relaxation of CSF acutely. Ultra- As experience was gained, subsequent reports (3, 4) indicated violet visible spectroscopy of bloody CSF that acute intracranial hemorrhage could be much more difficult to stored hypoxically for 3 days showed the detect on MR images. This was attributed by Sipponen et al. (3) to a presence of methemoglobin. The iron in lack of Ti shortening during the acute phase; however, they at- methemoglobin is paramagnetic; in com- tempted no explanation of this phenomenon. They reported that bination with water this facilitates Ti re- acute subarachnoid hemorrhage was difficult to detect by MR laxation. It is concluded that methemo- study, particularly in comparison with computed tomography (CT) globin formation with Ti shortening at (4). Although DeLaPaz et ai. (4) agree that acute intracranial hemor- least partially accounts for the increasing rhage is difficult to detect on MR images, they disagree as to the intensity of the MR appearance of subar- mechanism causing the intense appearance. While the data of Sip- achnoid hemorrhage over time in the cen- ponen et al. (3), ‘Bailes et al. (1), and Bydder et al. (2) suggest a Ti- tral nervous system and may also explain shortening process, the data of DeLaPaz et a!. (4) suggest no change the intense appearance of subacute hem- in Ti but rather a prolongation of T2, both of which would increase orrhage in MR images elsewhere in the the intensity on spin-echo images. Figure 2 illustrates the rather body. subtle increased intensity present in acute subarachnoid hemor- rhage 17 hours after ictus. One week after the acute subarachnoid Index terms: Brain, hemorrhage #{149}Hemorrhage, hemorrhage, the appearance is significantly more intense (Fig. 3). magnetic resonance studies #{149}Red blood cells To attempt an understanding of the variable appearance of subar- achnoid hemorrhage on MR images, the structure of hemoglobin Radiology 1985; 156:99-103 and its various breakdown products must be considered in some detail. In its circulating form, hemoglobin alternates between the oxy and deoxy forms as oxygen is exchanged during its transit through the high-oxygen environment of the lungs and low-oxy- gen environment of the capillary circulation. To bind oxygen re- vensibly, the iron in the hemoglobin (heme iron) must be main- tamed in the reduced ferrous (Fe2) state (5). To do this, the red blood cell maintains several metabolic pathways to prevent various oxidizing agents from converting its heme iron to the nonfunc- tional fernic (Fe3) state. When hemoglobin is removed from the circulation, these metabolic pathways fail and the hemoglobin mol- ecule begins to undergo oxidative denaturation. 1 From the MR Imaging Laboratory, Huntington The heme iron normally is suspended in a nonpolar crevice in Medical Research Institutes (W.G.B., P.G.S.) and the the center of the hemoglobin molecule. It is held in this position by Department of Radiology (W.G.B.), Huntington Me- morial Hospital, Pasadena, California. Presented at the a covalent bond with a histadine at the so-called F8 position of the 70th Scientific Assembly and Annual Meeting of the globin chain and by four planar hydrophobic van den Waals bonds Radiological Society of North America, Washington, with various nonpolar groups on the globin molecule. The group D.C., November 25-30, 1984. Received January 11, attache4 to the sixth coordination site of the heme iron varies. It is 1985; accepted and revision requested February 1 1; re- vision received March 11. occupied by molecular oxygen in oxyhemoglobin; it is vacant in C RSNA, 1985 deoxyhemoglobin (Fig. 4). As the oxidative denaturation of hemo- 99 globin proceeds, the ferrous heme ing the red blood cells; to do this, fresh Figure 1 iron is oxidized to the fernic state and venous blood was repeatedly passed methemoglobin is formed. The five through a 25-gauge needle before being mixed with CSF. The magnetic relaxation bonds to the globin molecule are un- times of oxy- and deoxyhemoglobin were changed; the sixth coordination site is compared by bubbling either oxygen or now occupied by either a water mole- nitrogen through fresh solutions of cule or a hydroxyl ion, depending on bloody CSF. Methemoglobin was pro- whether the methemoglobin is acidic duced by treatment of deoxyhemoglobin or basic. At physiologic pH, the acid with sodium nitrite (NaNO2). Before mea- form predominates. With continued surement, all samples were agitated to sus- oxidative denaturation, methemoglo- pend the red cells. All measurements were bin is converted to derivatives known performed at 38#{176}C. as hemichromes (5). While the iron in Quantitation of subarachnoid hemor- rhage generally is performed by means of these compounds remains in the fer- light (visible) spectroscopy (8). Such anal- nc state, alteration of the tertiary ysis provides the “xanthochromic” index structure of the globin molecule oc- that is used to quantitate the degree of curs, with the result that the sixth co- hemorrhage. The xanthochromic index is ordination site of the heme iron is oc- the sum of the absorption values at 415 nm cupied by a ligand from within the (oxyhemoglobin) and 460 nm (bilirubin). Subacute subarachnoid hemorrhage. Intra- globin molecule (most likely the dis- All samples are prepared for spectropho- parenchymal hematoma (arrow) and subar- tal histadine at the E7 position). tometric analysis by centrifugation, with achnoid hemorrhage (arrowhead) are noted To consider the effect of subarach- examination of only the supernatant. By 1 week after rupture of aneurysm in anterior such analysis, little methemoglobin has noid hemorrhage on MR imaging communicating artery. The contrast between previously been found either in acute su- the lesions and the surrounding brain is en- over time, one must evaluate the in- barachnoid hemorrhage or within several hanced on this Ti-weighted spin-echo image tenaction between the iron-contain- weeks after the hemorrhage (8, 9). (TR = 0.5 sec; TE 28 msec). ing hemoglobin within the red blood cells and water protons in the cere- brospinal fluid (CSF). A trivial expla- nation for the Ti shortening apparent on images of subacute hemorrhage might be the lysis of red blood cells that can occur as a result of their expo- sure to phospholipases in the CSF. We can readily exclude this mechanism for enhanced proton relaxation be- cause water molecules are already al- lowed access to the hemoglobin by rapid transit across the red cell mem- brane (6, 7). Thus, changes in proton relaxation enhancement effects may be attributed directly to changes in the interactions between the CSF wa- ten protons and the heme iron that occur as a result of oxidative denatun- ation of hemoglobin. In view of the discrepant reports in the MR imaging literature and the lack of an obvious explanation from a. b. the literature on MR and ultraviolet- Acute subarachnoid hemorrhage 17 hours post ictus. a. Midline sagittal section shows minimally increased intensity in the pontine and medul- visible (UV) spectroscopy for the lary cisterns (arrowheads). The intensity of the CSF in these subarachnoid spaces should changing appearance of subarach- . be the same as that in the fourth ventricle (arrow). noid hemorrhage, the following stud- b. Coronal section demonstrates minimally increased intensity in the left sylvian cistern on ies were undertaken. this TR 1.5-sec. TE 28-msec image. The solutions of oxy- and deoxyhemog- CSF) and compared with the known stan- lobin and methemoglobin were analyzed dards previously produced. MATERIALS AND METHODS using a Perkin-Elmer UV-visible spectro- photometer. Attention was directed to the Subarachnoid hemorrhage was mod- 630-nm region, which is specific for meth- RESULTS eled in vitro by adding fresh venous hu- emoglobin. man blood to artificial CSF, producing a To assess the change in magnetic relax- The effect of concentration of blood 10% (by volume) solution. The Ti and T2 ation times for bloody CSF stored hypoxi- in CSF is demonstrated in Figure 5, relaxation times of this solution were eval- cally, a 10% solution of lysed red blood which shows a small (10%) decrease uated using an IBM Minispec desktop cells in CSF was measured over several in Ti and T2 relaxation times as the spectrometer operating at 20 MHz. The ef- days. In a second experiment, a 20% solu- concentration is increased from 0% fect of red blood cell concentration on Ti tion of whole red cells in CSF was again (pure CSF) to 10% red blood cells. and T2 relaxation was evaluated by mea- stored hypoxically for several days with suring the relaxation times at concentra- sequential measurement of Ti and T2 re- Such Ti shortening cleanly is not the tions varying from 0% (pure CSF) to 10% laxation times.