Glucose-6-phosphate Dehydrogenase Testing Research Articles

Establishing a unique specimen repository with characterized G6PD enzyme activity for the development of new G6PD point-of-care tests

Glucose-6-phosphate dehydrogenase (G6PD) deficiency tests play a critical role in supporting the safe treatment and elimination of malaria. Evaluating G6PD activity in those infected with Plasmodium vivax will be essential in order to treat and achieve radical cures. Our goal is to establish a unique specimen repository with cryopreserved specimens highly characterized for G6PD activity. Providing such specimens to designers of G6PD point-of-care tests, will support both the development and evaluation of new diagnostic devices. Because G6PD-deficiency prevalence ranges from 15%, obtaining fresh blood specimens with a range of levels of G6PD activity - as well as specimens from heterozygous females - typically requires constant screening from large numbers of subjects. Thus, developers would highly benefit by using the same panel of specimens from the repository over the entire course of the development of a specific G6PD test. Currently the repository constitutes specimens from adults of known G6PD-deficient population groups and will be expanded over time with diverse ethnic backgrounds. Standard operation procedures and formulations have been optimized to allow cryopreservation of red blood cells (RBC) and stabilize G6PD enzyme activity within the RBC. Flow cytometry and kinetic assays demonstrate that specimens can be stored a minimum of six months under these optimized conditions. Critically, thawed specimens demonstrate stability for seven days. We are using Freezerworks database to maintain the chain of custody of all specimens. Cytochemical staining is performed to pedigree the specimens. The specimens are processed and tested for G6PD activity immediately by quantitative and qualitative assays. Samples are stored depending on the phenotype in order to increase the capacity of G6PD diverse specimens. Samples with normal G6PD activity are more prevalent and discarded to accommodate intermediate and deficient specimens. Once samples have been tested and selected for the repository, three preservation methods are used: (1) whole RBCs are cryopreserved in glycerolyte and stored in liquid nitrogen for G6PD phenotyping, (2) whole RBCs without glycerolyte are preserved in liquid nitrogen for phenotyping, and (3) peripheral mononuclear blood cells are preserved for DNA sequencing and genotyping. The G6PD specimen repository at PATH will provide a low-cost service to research, commercial, and other organizations with compatible product development goals that benefit the global health community.

Pharmacovigilance for single low-dose primaquine: a practical approach

Establishing and strengthening pharmacovigilance in resource-limited settings provides a valuable opportunity to identify, quantify and address pertinent safety concerns regarding the use of single low-dose (SLD) primaquine. While it is understood that primaquine causes dose-dependent hemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals with P. falciparum and P. vivax malaria, the extent of this risk when using low doses of primaquine remains to be defined. Prospective pharmacovigilance methods are needed to confirm or refute these safety concerns that have likely hindered the uptake of the new WHO policy for SLD primaquine. Pharmacovigilance in resource-limited settings is challenging, especially when attempting to identify unknown or poorly understood adverse drug reactions over weeks of follow-up. In contrast, the objectives of a pharmacovigilance program for SLD primaquine are specific, measurable, and attainable within a short time frame. The nadir of primaquine-induced hemolytic anemia, measurable by hemoglobin concentrations, has been shown to occur on or near day 7. Thus, it is expected that adverse drug reactions related to primaquine can be captured within a week of drug administration by active surveillance methods involving patient follow-up. Furthermore, dark urine is a characteristic sign of acute hemolytic anemia, an easily identifiable and documentable symptom of hemolysis. Numerous pharmacovigilance efforts are ongoing, supporting individual countries in the collection of standardized SLD primaquine safety data. These efforts seek to establish the expected fall in hemoglobin from baseline levels before treatment to levels seven days post-treatment and include G6PD testing and recording of adverse events using standard data collection instruments. Each participating in-country program or study site aims to collect data on 100-400 subjects depending on malaria endemicity and expected G6PD prevalence. We plan to collate data across these countries in a global database of individual patient data, contributing to the evidence-base for benefit-risk assessments of SLD primaquine. As other 8-aminoquinoline drugs such as tafenoquine come to market, we will be faced with related safety concerns of dose-dependent hemolysis in G6PD-deficient individuals. Active pharmacovigilance and the establishment of a standardized database for SLD primaquine not only provide opportunities to harmonize efforts, address unanswered questions regarding the safe use of SLD primaquine, and assist programs planning widespread roll-out of SLD primaquine in routine malaria case management they can also serve as a common platform for other current and future pharmacovigilance needs. The methods used in these efforts and the composition of this database will be described.

First evaluation of glucose-6-phosphate dehydrogenase (G6PD) deficiency in vivax malaria endemic regions in the Republic of Korea.

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK).MethodsTwo commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK.ResultsOf 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants.ConclusionsNo G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.

Performance of the CareStart glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test in Gressier, Haiti.

Administering primaquine (PQ) to treat malaria patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency can pose a serious risk of drug-induced hemolysis (DIH). New easy to use point-of-care rapid diagnostic tests are being developed as an alternative to labor-intensive spectrophotometric methods, but they require field testing before they can be used at scale. This study screened 456 participants in Gressier, Haiti using the Access Bio CareStart qualitative G6PD rapid detection test compared with the laboratory-based Trinity Biotech quantitative spectrophotometric assay. Findings suggest that the CareStart test was 90% sensitive for detecting individuals with severe deficiency and 84.8% sensitive for detecting individuals with moderate and severe deficiency compared with the Trinity Biotech assay. A high negative predictive value of 98.2% indicates excellent performance in determining those patients able to take PQ safely. The CareStart G6PD test holds much value for screening malaria patients to determine eligibility for PQ therapy.

Mass primaquine treatment to eliminate vivax malaria: lessons from the past.

Recent successes in malaria control have put malaria eradication back on the public health agenda. A significant obstacle to malaria elimination in Asia is the large burden of Plasmodium vivax, which is more difficult to eliminate than Plasmodium falciparum. Persistent P. vivax liver stages can be eliminated only by radical treatment with a ≥ seven-day course of an 8-aminoquinoline, with the attendant risk of acute haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Primaquine is the only generally available 8-aminoquinoline. Testing for G6PD deficiency is not widely available, and so whilst it is widely recommended, primaquine is often not prescribed. In the past, some countries aiming for vivax malaria eradication deployed mass treatments with primaquine on a massive scale, without G6PD testing. In Azerbaijan, Tajikistan (formerly USSR), North Afghanistan and DPR Korea 8,270,185 people received either a 14-day “standard” or a 17-day “interrupted” primaquine treatment to control post-eradication malaria epidemics. These mass primaquine preventive treatment campaigns were conducted by dedicated teams who administered the drugs under supervision and then monitored the population for adverse events. Despite estimated G6PD prevalences up to 38.7%, the reported frequency of severe adverse events related to primaquine was very low. This experience shows that with careful planning and implementation of mass treatment strategies using primaquine and adequate medical support to manage haemolytic toxicity, it is possible to achieve high population coverage, substantially reduce malaria transmission, and manage the risk of severe acute haemolytic anaemia in communities with a relatively high prevalence of G6PD deficiency safely.

Screening and prevention of neonatal glucose 6-phosphate dehydrogenase deficiency in Guangzhou, China.

We aimed to summarize the results of screening protocol and prevention of neonatal glucose 6-phosphate dehydrogenase (G6PD) deficiency during a 22-year-long period to provide a basis of reference for the screening of this disease. About 1,705,569 newborn subjects in Guangzhou City were screened for this deficiency. Specimens were collected according to the conventional method of specimen acquisition for "newborn dried bloodspot screening", preserved, and inspected. The specimens were studied with fluorescent spot test and quantitative fluorescence assay. Diagnosis was performed using the modified NBTG6PD/6PGD ratio method. Bloodspot filter paper specimens were sent to the laboratory within 24 h via EMS Express, and the G6PD test was performed on the same day. The G6PD deficiency-positive rate was 4.2% in the samples screened using the fluorescent spot test, while it was 5% in case of the quantitative fluorescence assay. Neonatal screening for G6PD deficiency for 11,437 cases (6117 boys and 5320 girls) showed positive results in 481 cases. About 420 cases (318 boys and 102 girls) of G6PD deficiency were confirmed with the modified Duchenne NBT ratio method. The total detection rate was 3.7:5.2% for boys and 1.9% for girls. Quantitative fluorescence assay improved the sensitivity and detection rate. Accelerating the speed of sample delivery by using Internet network systems and ensuring online availability of screening results can aid the screening and diagnosis of this deficiency within 1 week of birth.

G6PD deficiency: a classic example of pharmacogenetics with on-going clinical implications.

That primaquine and other drugs can trigger acute haemolytic anaemia in subjects who have an inherited mutation of the glucose 6-phosphate dehydrogenase (G6PD) gene has been known for over half a century: however, these events still occur, because when giving the drug either the G6PD status of a person is not known, or the risk of this potentially life-threatening complication is under-estimated. Here we review briefly the genetic basis of G6PD deficiency, and then the pathophysiology and the clinical features of drug-induced haemolysis; we also update the list of potentially haemolytic drugs (which includes rasburicase). It is now clear that it is not good practice to give one of these drugs before testing a person for his/her G6PD status, especially in populations in whom G6PD deficiency is common. We discuss therefore how G6PD testing can be done reconciling safety with cost; this is once again becoming of public health importance, as more countries are moving along the pathway of malaria elimination, that might require mass administration of primaquine. Finally, we sketch the triangular relationship between malaria, antimalarials such as primaquine, and G6PD deficiency: which is to some extent protective against malaria, but also a genetically determined hazard when taking primaquine.

G6PD testing in support of treatment and elimination of malaria: recommendations for evaluation of G6PD tests

Malaria elimination will be possible only with serious attempts to address asymptomatic infection and chronic infection by both Plasmodium falciparum and Plasmodium vivax. Currently available drugs that can completely clear a human of P. vivax (known as “radical cure”), and that can reduce transmission of malaria parasites, are those in the 8-aminoquinoline drug family, such as primaquine. Unfortunately, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency risk having severe adverse reactions if exposed to these drugs at certain doses. G6PD deficiency is the most common human enzyme defect, affecting approximately 400 million people worldwide.Scaling up radical cure regimens will require testing for G6PD deficiency, at two levels: 1) the individual level to ensure safe case management, and 2) the population level to understand the risk in the local population to guide Plasmodium vivax treatment policy. Several technical and operational knowledge gaps must be addressed to expand access to G6PD deficiency testing and to ensure that a patient’s G6PD status is known before deciding to administer an 8-aminoquinoline-based drug.In this report from a stakeholder meeting held in Thailand on October 4 and 5, 2012, G6PD testing in support of radical cure is discussed in detail. The focus is on challenges to the development and evaluation of G6PD diagnostic tests, and on challenges related to the operational aspects of implementing G6PD testing in support of radical cure. The report also describes recommendations for evaluation of diagnostic tests for G6PD deficiency in support of radical cure.

Mortality Following Rasburicase-Induced Methemoglobinemia

To report a case of mortality following rasburicase-induced methemoglobinemia. A 62-year-old African American male with chronic lymphocytic leukemia and small lymphocytic lymphoma was admitted for tumor lysis syndrome and renal failure. He was treated with 2 doses of rasburicase, subsequently developed methemoglobinemia, and required intubation, multiple packed red blood cell (PRBC) transfusions, and 2 doses of methylene blue. A screen for glucose-6 phosphate dehydrogenase (G6PD) deficiency was negative. His course was complicated by hemolytic anemia, nosocomial pneumonia, Clostridium difficile infection, and septic shock. His methemoglobin concentrations normalized over several days; however, the patient eventually died on hospital day 16. An objective causality assessment revealed that the adverse drug reaction was probable. Our case was similar to previously published cases, except that our patient died and his G6PD screen was negative. Although it was negative, it is likely that this was a false negative result because this blood was drawn shortly after PRBC transfusions and during active hemolysis. Both these are likely to cause false-negative results. Methemoglobinemia is a rare adverse effect associated with the use of rasburicase and occurs most often in patients with G6PD deficiency. G6PD testing should not be ordered during active hemolysis or after blood transfusion because this may lead to false-negative results. Methylene blue should not be used as an antidote because it may worsen hemolytic anemia in patients with G6PD deficiency.

Lower reference limits of quantitative cord glucose-6-phosphate dehydrogenase estimated from healthy term neonates according to the Clinical and Laboratory Standards Institute guidelines: a cross sectional retrospective study.

BackgroundPrevious studies have reported the lower reference limit (LRL) of quantitative cord glucose-6-phosphate dehydrogenase (G6PD), but they have not used approved international statistical methodology. Using common standards is expecting to yield more true findings. Therefore, we aimed to estimate LRL of quantitative G6PD detection in healthy term neonates by using statistical analyses endorsed by the International Federation of Clinical Chemistry (IFCC) and the Clinical and Laboratory Standards Institute (CLSI) for reference interval estimation.MethodsThis cross sectional retrospective study was performed at King Abdulaziz Hospital, Saudi Arabia, between March 2010 and June 2012. The study monitored consecutive neonates born to mothers from one Arab Muslim tribe that was assumed to have a low prevalence of G6PD-deficiency. Neonates that satisfied the following criteria were included: full-term birth (37 weeks); no admission to the special care nursery; no phototherapy treatment; negative direct antiglobulin test; and fathers of female neonates were from the same mothers’ tribe. The G6PD activity (Units/gram Hemoglobin) was measured spectrophotometrically by an automated kit. This study used statistical analyses endorsed by IFCC and CLSI for reference interval estimation. The 2.5th percentiles and the corresponding 95% confidence intervals (CI) were estimated as LRLs, both in presence and absence of outliers.Results207 males and 188 females term neonates who had cord blood quantitative G6PD testing met the inclusion criteria. Method of Horn detected 20 G6PD values as outliers (8 males and 12 females). Distributions of quantitative cord G6PD values exhibited a normal distribution in absence of the outliers only. The Harris-Boyd method and proportion criteria revealed that combined gender LRLs were reliable. The combined bootstrap LRL in presence of the outliers was 10.0 (95% CI: 7.5-10.7) and the combined parametric LRL in absence of the outliers was 11.0 (95% CI: 10.5-11.3).ConclusionThese results contribute to the LRL of quantitative cord G6PD detection in full-term neonates. They are transferable to another laboratory when pre-analytical factors and testing methods are comparable and the IFCC-CLSI requirements of transference are satisfied. We are suggesting using estimated LRL in absence of the outliers as mislabeling G6PD-deficient neonates as normal is intolerable whereas mislabeling G6PD-normal neonates as deficient is tolerable.

Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in Uganda

BackgroundThe distribution of the enzymopathy glucose-6-phosphate dehydrogenase (G6PD) deficiency is linked to areas of high malaria endemicity due to its association with protection from disease. G6PD deficiency is also identified as the cause of severe haemolysis following administration of the anti-malarial drug primaquine and further use of this drug will likely require identification of G6PD deficiency on a population level. Current conventional methods for G6PD screening have various disadvantages for field use.MethodsThe WST8/1-methoxy PMS method, recently adapted for field use, was validated using a gold standard enzymatic assay (R&D Diagnostics Ltd ®) in a study involving 235 children under five years of age, who were recruited by random selection from a cohort study in Tororo, Uganda. Blood spots were collected by finger-prick onto filter paper at routine visits, and G6PD activity was determined by both tests. Performance of the WST8/1-methoxy PMS test under various temperature, light, and storage conditions was evaluated.ResultsThe WST8/1-methoxy PMS assay was found to have 72% sensitivity and 98% specificity when compared to the commercial enzymatic assay and the AUC was 0.904, suggesting good agreement. Misclassifications were at borderline values of G6PD activity between mild and normal levels, or related to outlier haemoglobin values (<8.0 gHb/dl or >14 gHb/dl) associated with ongoing anaemia or recent haemolytic crises. Although severe G6PD deficiency was not found in the area, the test enabled identification of low G6PD activity. The assay was found to be highly robust for field use; showing less light sensitivity, good performance over a wide temperature range, and good capacity for medium-to-long term storage.ConclusionsThe WST8/1-methoxy PMS assay was comparable to the currently used standard enzymatic test, and offers advantages in terms of cost, storage, portability and use in resource-limited settings. Such features make this test a potential key tool for deployment in the field for point of care assessment prior to primaquine administration in malaria-endemic areas. As with other G6PD tests, outlier haemoglobin levels may confound G6PD level estimation.

Should we screen newborns for glucose-6-phosphate dehydrogenase deficiency in the United States?

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common X-linked enzymopathy can lead to severe hyperbilirubinemia, acute bilirubin encephalopathy and kernicterus in the United States. Neonatal testing for G6PD deficiency is not yet routine and the American Academy of Pediatrics recommends testing only in jaundiced newborns who are receiving phototherapy whose family history, ethnicity, or geographic origin suggest risk for the condition, or for infants whose response to phototherapy is poor. Screening tests for G6PD deficiency are available, are suitable for use in newborns and have been used in birth hospitals. However, US birth hospitals experience is limited and no national consensus has emerged regarding the need for newborn G6PD testing, its effectiveness or the best approach. Our review of current state of G6PD deficiency screening highlights research gaps and informs specific operational challenges to implement universal newborn G6PD testing concurrent to bilirubin screening in the United States.

Quantitative Neonatal Glucose-6-Phosphate Dehydrogenase Screening: Distribution, Reference Values, and Classification by Phenotype

To determine enzyme assay reference values for newborns in a Sephardic Jewish population at high risk for glucose-6-phosphate dehydrogenase (G6PD) deficiency. Quantitative G6PD testing was performed on umbilical cord blood. The reduction of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate-oxidase, reflecting G6PD activity, was measured spectrophotometrically. Hemoglobin (Hb) was measured on the same sample. G6PD activity was recorded as U/g Hb. Males (N = 1502) were separated into 2 distinct groups: those <7 U/g Hb (n = 243 [16.2%], median 0.28 U/g Hb), designated G6PD deficient, presumably hemizygotes; and those ≥ 9 U/g Hb (n = 1256 [83.8%], 18.76 U/g Hb), designated G6PD normal, presumably hemizygotes. Female (n = 1298) values were a continuum and were categorized based on the male distribution: those <7 U/g Hb (n = 81 [6.2%], 4.84 U/g Hb), G6PD deficient, probably homozogytes; those ≥ 9.5 U/g Hb, equivalent to 50% of the male normal value, (n = 1153 (88.8%), 18.36 U/g Hb), G6PD normal, probably homozygotes; and those with intermediate values (n = 64 [4.9%], 8.61 U/g Hb), probable heterozygotes. Accurate identification of the male G6PD-deficient state was possible despite high normal neonatal G6PD values. Female values were presented as a continuum preventing accurate classification but were classified based on male phenotype for practical use.

Prevalence of G6PD deficiency in a large cohort of HIV-infected patients

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. Screening for this condition in HIV-infected patients from susceptible ethnic groups is recommended based on expert opinion. Here we determined the prevalence of G6PD deficiency and the occurrence of G6PD-related hemolytic events in a large cohort of patients. We identified all HIV-infected adults who presented as new patients at a single urban HIV clinic between 02/01/2007 and 01/31/2009. Demographic and laboratory data including G6PD results were collected. In addition, outpatient and inpatient medical records of G6PD deficient patients were reviewed for episodes of hemolytic anemia. A total of 1172 patients were identified and G6PD testing was performed in 1110 (94.7%). Overall, 75 (6.8%) subjects had G6PD deficiency. Rates were higher among African Americans (68/699; 9.7%) and Hispanics (5/253; 2.0%). Only one non-Hispanic White subject had G6PD deficiency (1/153; 0.7%). At baseline, hemoglobin concentrations were similar among subjects with or without G6PD deficiency. Among patients with G6PD deficiency, 40 (53.3%) were prescribed trimethoprim-sulfamethoxazole or dapsone. During follow-up, five (6.7%) of these patients developed acute hemolytic anemia. These results provide strong clinical evidence for recommending G6PD testing in HIV-infected subjects from susceptible ethnic backgrounds.

A Randomised Trial of an Eight-Week, Once Weekly Primaquine Regimen to Prevent Relapse of Plasmodium vivax in Northwest Frontier Province, Pakistan

BackgroundVivax malaria remains a major cause of morbidity in the subtropics. To undermine the stability of the disease, drugs are required that prevent relapse and provide reservoir reduction. A 14-day course of primaquine (PQ) is effective but cannot safely be used in routine practice because of its interaction with glucose-6-phosphate dehydrogenase (G6PD) deficiency for which testing is seldom available. Safe and effective use of PQ without the need for G6PD testing would be ideal. The efficacy and safety of an 8-week, once weekly PQ regimen was compared with current standard treatment (chloroquine alone) and a 14-day PQ regimen.Methods and Principal Findings200 microscopically confirmed Plasmodium vivax patients were randomly assigned to either once weekly 8-week PQ (0.75mg/kg/week), once weekly 8-week placebo, or 14-day PQ (0.5mg/kg/day) in North West Frontier Province, Pakistan. All patients were treated with a standard chloroquine dose and tested for G6PD deficiency. Deficient patients were assigned to the 8-week PQ group. Failure was defined as any subsequent episode of vivax malaria over 11 months of observation. There were 22/71 (31.0%) failures in the placebo group and 1/55 (1.8%) and 4/75 (5.1%) failures in the 14-day and 8-week PQ groups, respectively. Adjusted odds ratios were: for 8-week PQ vs. placebo-0.05 (95%CI: 0.01-0.2, p<0.001) and for 14-day PQ vs. placebo-0.01 (95%CI: 0.002-0.1, p<0.001). Restricted analysis allowing for a post-treatment prophylactic effect confirmed that the 8-week regimen was superior to current treatment. Only one G6PD deficient patient presented. There were no serious adverse events.ConclusionsA practical radical treatment for vivax malaria is essential for control and elimination of the disease. The 8-week PQ course is more effective at preventing relapse than current treatment with chloroquine alone. Widespread use of the 8-week regimen could make an important contribution to reservoir reduction or regional elimination where G6PD testing is not available.Trial RegistrationClinicalTrials.gov NCT00158587

Hepatitis E Virus Infection with Nonimmune Hemolytic Anemia

Hepatitis E Virus Infection with Nonimmune Hemolytic Anemia

Histochemical studies on the distribution of thiamine pyrophosphatase and enzymes related to carbohydrate metabolism in the intercalated neurons of the rat supraoptic nucleus.

Histochemical studies have been conducted by applying hexokinase (HK), aldolase (AD), glyceraldehyde-3-phosphate dehydrogenase (G3), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PD), and thiamine pyrophosphatase (TPPase) methods, as well as Nissl staining and Gomori's chrome-alum-hematoxylin-phloxine (CHP) methods to intercalated neurons of the supraoptic nucleus (SO) on Wistar strain rats. Intercalated neurons reacted weakly to the AD, G3, G6PD, and SDH tests, indicating that they belong to the category of ordinary neurons with low carbohydrate metabolism. Many fibrous astrocytes showing strong HK reactions surround neurosecretory neurons. However, they do not surround intercalated neurons with mild HK activity. These results indicate that the latter receive a poor supply of energy from glucose in the circulating blood in contrast to the former. Intercalated neurons are very rich in Nissl substance but lack CHP-positive material. They may have a high potential for synthesizing protein. The principal morphological features of the TPPase-positive Golgi material are peculiar and heterogeneous shape and poor development. These findings together with mild G6PD activity suggest that intercalated neurons are very likely to have poor synthesizing activity.