Development, diagnosis and therapy of ketosis in non-gravid and non-lactating Guinea pigs.

BACKGROUND: Ketosis is a metabolic disorder often triggered by anorexia in animals fed on high energy diets. Although mostly described in pregnant female guinea pigs, under the name of pregnancy toxicosis; there is limited information on ketosis in males and non-pregnant females, often presented to clinics with anorexia or inappetence. The objective of this study was to observe progression of ketosis in guinea pigs, document the changes and evaluate diagnostic methods and a therapeutic approach. RESULTS: Twenty eight adult guinea pigs (Cavia porcellus), castrated males and intact females of obese and slim body condition were fasted for 3 days and refed afterwards. The slim animals served as control group for body condition. Either slim and fat animals were divided into two treatment groups: half of them received fluid replacements with glucose subcutaneously, the other half did not receive any injection and served as treatment control. Serum beta-hydroxybutyrate, and urine acetoacetate and acetone were measured during and after fasting. Serum ALT, bile acids and liver histology were also analyzed after 7 days of refeeding (and therapy). Females and obese guinea pigs showed a significantly higher increase in ketone bodies in serum and urine. Obese, female, or animals not receiving therapy needed more time to regulate ketone bodies to normal levels than slim animals, males or animals receiving therapy. Liver histology revealed increased hepatocyte degeneration and higher glycogen content in obese animals and animals receiving therapy, and additionally more glycogen content in males. Only minor hepatic fat accumulation was documented. Bile acids showed good correlation to histological liver changes whereas ALT did not. CONCLUSIONS: Female and obese animals react more intensively to fasting. As preventive management, animals should be kept in adequate body condition, fasting should be avoided, and anorexia should be treated immediately. In such a case, urinary dip sticks to detect ketone bodies are a useful diagnostic tool. Glucose therapy leads to faster cessation of ketogenesis and should be recommended in cases of ketosis. However, it needs to be adjusted to avoid hepatocyte glycogen overload and degeneration. Measuring bile acids presents a valuable indicator of liver damage.

[Overview on the relationship between changes in activity and ketosis in dairy cows]. / Übersicht zu Zusammenhängen zwischen Änderungen der Bewegungsintensität und Ketose bei Milchkühen.

With a prevalence of up to 43 % subclinical ketosis is one of the most common diseases in dairy cows in their transition period. In itself, this may cause subsequent diseases such as clinical ketosis or lameness. Therefore, monitoring of animals in this stage is of importance. In addition to the measurement of ß-hydroxybutyrate or acetoacetate in blood, milk, and urine as well as the observation of the animals, computer-assisted systems are suitable means of monitoring. Information such as animal identification and activity data are recorded on a data logger and transmitted to a computer. A change in activity may be an indication of an underlying disease days before the onset of additional clinical signs. In cases of ketosis, a decrease in activity may be observed 5 days before the clinical diagnosis is made. Thus, these data are a valuable contribution in monitoring the cattle herd's health status for both the farmer and the veterinarian. Activity measurement may also be employed for the detection of a beginning lameness. In the presence of lameness, the individual's activity decreases and periods of lying are longer. Activity measurement via transponder as a part of the herd monitoring provides important information on lameness prevalence in the herd. In the presence of a lameness a visual assessment should additionally be made. Lameness scores (Locomotion score, Gait score) have been developed for this purpose and add to determining the lameness status of the herd. This way the animals are divided into different lameness classes. Based on this classification those individuals in need of claw trimming or further treatment may be identified leading to amelioration or prevention of secondary diseases. Due to lameness and subsequent reduction of activity and feed intake, the animals may develop subclinical or clinical ketosis. Therefore, under consideration of both animal welfare and economic factors early disease detection and prophylaxis is desirable and should be a main objective of herd monitoring.

Comparison of Intermittent Fasting Versus Caloric Restriction in Obese Subjects: A Two Year Follow-Up.

OBJECTIVE: Caloric restriction (CR) is proven to be effective in increasing life span and it is well known that, nutritional habits, sleeping pattern and meal frequency have profound effects on human health. In Ramadan some Muslims fast during the day-light hours for a month, providing us a unique model of intermittent fasting (IF) in humans. In the present study, we have investigated the effects of IF versus CR on the same non-diabetic obese subjects who were followed for two years according to the growth hormone (GH)/Insulin like growth factor (IGF)-1 axis and insulin resistance. DESIGN: Single-arm Interventional Human Study. PARTICIPANTS: 23 female subjects (Body Mass Index (BMI) 29-39, aged between 28-42years). SETTING: Follow-up is designed as 12 months of CR, after which there was a month of IF and 11 months of CR again, to be totally 24 months. Subjects' daily diets were aligned as low calorie diet during CR and during the IF period, the same subjects fasted for 15 hours in a day for a month and there was no daily calorie restriction. Nutritional pattern was changed as 1 meal in the evening and a late supper before sleeping and no eating and drinking during the day light hours in the IF model. Subjects made brisk walking twice a day during the whole follow-up including both CR and IF periods. BMI, Blood glucose, insulin, TSH, GH, HbA1c, IGF-1, Homa-IR and urinary acetoacetate levels were monitored once in three months and twice in the fasting month. MEASUREMENTS AND RESULTS: While subjects lost 1250 ± 372g monthly during the CR, in the IF period, weight loss was decreased to 473 ± 146 g. BMI of all subjects decreased gradually and as the BMI decreased, glucose, HbA1c, insulin, Homa-IR and TSH levels were decreased. GH levels were at baseline at the beginning, increased in the first six months and stayed steady during the CR and IF period than began decreasing after the IF period, while IGF-I increased gradually during the CR period and beginning with the 7th day of IF period, it decreased and kept on decreasing till the end of the follow-up. Urinary acetoacetate levels were higher during the IF period suggesting a constant lipid catabolism. CONCLUSION: Our results suggest that, CR affects metabolic parameters positively which will help especially pre-diabetic and insulin resistant patients without any pharmacological approach. In addition IF without calorie restriction can enhance health and cellular resistance to disease without losing weight and those effects may be attributed to different signalling pathways and circulating ketones during IF. Changes observed during IF are probably due to the changes in eating and sleeping pattern and thus changes in metabolic rhythm.

Blood ß-hydroxybutyrate vs. urine acetoacetate testing for the prevention and management of ketoacidosis in Type 1 diabetes: a systematic review.

AIM: Diabetic ketoacidosis is a life-threatening complication of Type 1 diabetes. Blood ß-hydroxybutyrate testing is now widely available as an alternative to urine acetoacetate testing for detecting ketosis. The aim of this study was to review the effectiveness of capillary or serum ß-hydroxybutyrate compared with urine acetoacetate testing in prevention and management of diabetic ketoacidosis. METHODS: MEDLINE, EMBASE, EBM Reviews, The Cochrane Library and CINAHL (until April 2012, no language restrictions, studies in humans) were searched for experimental and observational studies comparing the effectiveness of blood ß-hydroxybutyrate and urine acetoacetate testing. Outcomes examined were prevention of diabetic ketoacidosis, time to recovery from diabetic ketoacidosis, healthcare costs and patient or caregiver satisfaction. Additional sources included reference lists, conference proceedings and contact with experts in the field. RESULTS: Four studies (two randomized controlled trials and two cohort studies) met eligibility criteria, including 299 participants across 11 centres. Risk of bias was low to moderate. Blood ketone testing compared with urine testing was associated with reduced frequency of hospitalization (one study), reduced time to recovery from diabetic ketoacidosis (three studies), cost benefits (one study) and greater satisfaction (one study, intervention group only). No study assessed prevention of diabetic ketoacidosis. Meta-analysis could not be performed because of heterogeneity in study design and published data. CONCLUSIONS: There is evidence suggesting that blood ß-hydroxybutyrate testing is more effective than urine acetoacetate testing in reducing emergency department assessment, hospitalization and time to recovery from diabetic ketoacidosis, as well as potentially lowering healthcare expenditure. Further research in both young people and adults is needed.

Repeated intoxication presenting with azotemia, elevated serum osmolal gap, and metabolic acidosis with high anion gap: differential diagnosis, management, and prognosis.

A man with a history of alcoholism presented on two different occasions with mental changes, clinical signs of volume depletion, elevated serum osmolal gap, metabolic acidosis with high anion gap, metabolic alkalosis, hyponatremia, and azotemia after binge drinking of only ethanol. In both episodes, the serum contained ethanol, acetone, and 2-propanol (isopropanol), but no methanol or ethylene glycol. In the first episode, the rates of excretion of acetoacetate and 3-hydroxybutyrate in the urine were greatly increased. Volume repletion was the only treatment. In both episodes, azotemia and metabolic acidosis were rapidly reversed, while modest metabolic alkalosis was noted after treatment. The triad of azotemia, elevated osmolal gap, and high anion gap metabolic acidosis, which characterizes intoxication with methanol or ethylene glycol, can also develop in alcoholic ketoacidosis (AKA), an entity with substantially different management and outcome. Finding 2-propanol in the serum of patients with AKA indicates either concomitant 2-propanol ingestion or formation of 2-propanol from acetone.

Latent class evaluation of a milk test, a urine test, and the fat-to-protein percentage ratio in milk to diagnose ketosis in dairy cows.

In this study, 3 commonly used tests to diagnose ketosis were evaluated with a latent class model to avoid the assumption of an available perfect test. The 3 tests were the KetoLac BHB (Sanwa Kagaku Kenkyusho Co. Ltd., Nagoya, Japan) test strip that tests milk for ß-hydroxybutyrate, the KetoStix (Bayer Diagnostics Europe Ltd., Dublin, Ireland) test strip that tests urine for acetoacetate, and the fat-to-protein percentage ratio (FPR) in milk. A total of 8,902 cows were included in the analysis. The cows were considered to be a random sample from the population of Danish dairy cattle under intensive management, thus representing a natural spectrum of ketosis as a disease. All cows had a recorded FPR between 7 and 21 d postpartum. The KetoLac BHB recordings were available from 2,257 cows and 6,645 cows had a KetoStix recording. The recordings were analyzed with a modified Hui-Walter model, in a Bayesian framework. The specificity of the KetoLac BHB test and the KetoStix test were both high [0.99 (0.97-0.99)], whereas the specificity of FPR was somewhat lower [0.79 (0.77-0.81)]. The best sensitivity was for the KetoStix test [0.78 (0.55-0.98)], followed by the FPR [0.63 (0.58-0.71)] and KetoLac BHB test [0.58 (0.35-0.93)].

1H-nuclear magnetic resonance spectroscopy-based metabolic assessment in a rat model of obesity induced by a high-fat diet.

Obesity, whose prevalence is increasing rapidly worldwide, is recognized as a risk factor for diabetes, cardiovascular disease, liver disease, and renal disease. To investigate metabolic changes in the urine of a rat model of obesity induced by a high-fat diet (HFD), rats were divided into the following four groups based on the diet type and degree of weight gain: normal-diet (ND) low gainers, ND high gainers, HFD low gainers, and HFD high gainers. Biochemical analyses of visceral fat-pad weight, plasma, and liver tissues were performed. The (1)H-nuclear magnetic resonance ((1)H-NMR) spectra of urine were analyzed using multivariate statistical analysis to identify the separation of the groups. It was observed that the metabolic profile of urine obtained by (1)H-NMR-spectroscopy-based metabolomic analysis differed between ND low gainers and ND high gainers even though these animals consumed the same normal diet. Several key metabolites in urine, such as betaine, taurine, acetone/acetoacetate, phenylacetylglycine, pyruvate, lactate, and citrate contributed to the classification of these two groups. The metabolic profile of urine also differed between ND low gainers and HFD high gainers, which consumed the different diet and showed a different weight gain. This study has identified features of urine metabolites in various groups and demonstrated the reliability of an NMR-based metabolomics approach to investigate the effects of the diet and the physical constitution on obesity.

Bicarbonate therapy in severe metabolic acidosis.

The utility of bicarbonate administration to patients with severe metabolic acidosis remains controversial. Chronic bicarbonate replacement is obviously indicated for patients who continue to lose bicarbonate in the ambulatory setting, particularly patients with renal tubular acidosis syndromes or diarrhea. In patients with acute lactic acidosis and ketoacidosis, lactate and ketone bodies can be converted back to bicarbonate if the clinical situation improves. For these patients, therapy must be individualized. In general, bicarbonate should be given at an arterial blood pH of < or =7.0. The amount given should be what is calculated to bring the pH up to 7.2. The urge to give bicarbonate to a patient with severe acidemia is apt to be all but irresistible. Intervention should be restrained, however, unless the clinical situation clearly suggests benefit. Here we discuss the pros and cons of bicarbonate therapy for patients with severe metabolic acidosis.

Correlation between urine ketones (acetoacetate) and capillary blood ketones (3-beta-hydroxybutyrate) in hyperglycaemic patients.

AIMS: To facilitate the transition from urine ketones (acetoacetate) to capillary blood ketones (3-beta-hydroxybutyrate), we studied the correlation between these two tests. METHODS: Retrospective study of all patients with blood glucose greater than or equal to 2.5 g/l on arrival in the Emergency Department. We studied the correlation between urine ketones (Clinitek 50, Bayer) and capillary blood ketones (Optium, Abbott). We then compared the relative risks (RR) of ketoacidosis and hospitalization associated with each of these tests. RESULTS: In 33 months, 529 adult patients with both urine and blood testing for ketones were enrolled (ketoacidosis 8%, admission rate 49%). Urine ketones scored as +, ++ and +++ corresponded to median capillary blood ketone levels of 0.5 mmol/l (IQR: 0.1-0.9), 0.7 mmol/l (IQR: 0.2-1.8) and 3 mmol/l (IQR: 1.4-5.2), respectively. RRs of ketoacidosis or hospitalization associated with blood ketones greater than or equal to 3 mmol/l were higher than those associated with +++ urine ketones: 74 (95% confidence interval [CI]: 48-88) and 2.9 (95% CI: 2.5-3) versus 31 (95% CI: 18-45) and 2 (95% CI: 1.7-2.1), respectively. CONCLUSIONS: In hyperglycaemic patients in the Emergency Department, a good correlation was observed between urine ketones and capillary blood ketones for low values, but a poor correlation was observed for high values. Either test can therefore be used to exclude ketosis, but the capillary blood ketones test is more accurate to confirm ketoacidosis.

Blood ketone bodies in patients with recent-onset type 1 diabetes (a multicenter study).

BACKGROUND: Insulin deficiency with glucagon excess leads to the release of ketone bodies (KBs) by the liver and excretion in the urine. So far, only KB monitoring in urine has been used during assessment of children with diabetes. Currently used nitroprusside strips for urine KB detection react only with acetoacetate (AcAc) and not with the most prevalent KB moiety - 3beta-hydroxybutyrate (3HB) - that is in equilibrium with AcAc (up to 10:1 ratio). AIM: To verify the significance of 3HB in the blood compared to that of AcAc in the urine in recently diagnosed type 1 diabetic subjects independent of the presence of diabetic ketoacidosis (DKA). METHODS: A total of 118 consecutive newly diagnosed subjects with type 1 diabetes from different centers in Italy underwent intensive insulin therapy and hydration when necessary (38/118 = 32% with DKA). Hourly urine and capillary blood samples were used to monitor KBs until metabolic control was achieved. RESULTS: Although DKA was present in 32% of patients, blood 3HB was significantly increased (3.56 +/- 1.7 mmol/L) in 83% of the patients and correlated with blood glucose (r = 0.39; p < 0.01) according to a bimodal model. The strongest association was found between 3HB and venous pH (r =-0.56; p < 0.0001). Time required for blood 3HB normalization depended strongly on the starting blood KB values (r = 0.44; p < 0.0001) and was significantly lower than that required for disappearance of KB from urine (17.4 +/- 13.6 h, range 1-69 h vs. 19.7 +/- 17.8 h, range 1-120 h; p = 0.004). However, urine KBs disappeared before blood 3HB normalization in 23% of the patients. CONCLUSIONS: Blood 3HB evaluation is a better indicator of metabolic control compared to urine KB detection and is useful to predict the time required for blood KB clearing. Further studies are needed to assess its use in the early detection and management of DKA.

Prevention of fatty liver in transition dairy cows by subcutaneous injections of glucagon.

The main objective of this study was to test the extent to which injecting glucagon subcutaneously for 14 d beginning at d 2 postpartum would prevent fatty liver development in transition dairy cows. Twenty-four multiparous Holstein cows were fed 6 kg of cracked corn in addition to their standard diet during the last 30 d of a dry period to induce postpartum development of fatty liver. Glucagon at either 7.5 or 15 mg/d or saline (control) was injected subcutaneously 3 times daily for 14 d beginning at d 2 postpartum. Glucagon at 15 mg/ d prevented liver triacylglycerol accumulation in postpartum dairy cows. Glucagon at 7.5 mg/d showed potential for fatty liver prevention. Glucagon increased concentration of plasma glucose and insulin and decreased plasma nonesterified fatty acid concentrations. No effects of glucagon were detected on plasma beta-hydroxybutyrate concentrations. Glucagon affected neither feed intake nor milk production. Moreover, milk composition was not altered by glucagon. Milk urea N concentrations decreased, and plasma urea N concentrations tended to decrease during glucagon administration, indicating that glucagon may improve protein use. Liver glycogen concentrations were not affected by glucagon. No significant differences in body condition scores were detected among treatments throughout the study. These results indicate that subcutaneous glucagon injections can prevent fatty liver in transition dairy cows without causing major production and metabolite disturbances.

Quantitative measurement of ketone bodies in urine using reflectometry.

BACKGROUND: Recently, automated urine test strip readers became available that can report quantitative data. We explored the possibility of measuring all ketone bodies (acetone, acetoacetate, 3-hydroxybutyrate) in urine with these test strips. Monitoring urinary ketone concentrations could offer the advantages of measuring higher values (due to the low renal thresholds) and being less sensitive to fluctuations. METHODS: We evaluated URISYS 2400 (Roche) quantitative reflectance data for the ketone reflectance field and compared it with biochemical data from urine samples. Using an easy sample pre-treatment with 3-hydroxybutyrate dehydrogenase, we were able to assay 3-hydroxybutyrate as well, which normally does not react on urine test strips. RESULTS: Within- and between-run reproducibility of the reflectance signal for high- and low-concentration urine pools was 11.0-3.6% and 11.0-5.8% for aceto-acetate, 8.2-9.2% and 10.4-16.1% for acetone, and 5.1-3.0% and 5.6-3.5% for 3-hydroxybutyrate, respectively. The lower limit of detection for acetoacetate was 0.13 mmol/L (CV=3.6%). Fair agreement was obtained between test strip data for ketones andcolorimetrically determined acetoacetate values (r=0.90). CONCLUSIONS: In urine test strip analysis, quantitative ketone reflectance data allow a simple and fast analysis, offering affordable screening for the detection of ketone body production in diabetes, especially in emergency settings.

Evaluation and use of three cowside tests for detection of subclinical ketosis in early postpartum cows.

The objective was to evaluate the performance of 3 cowside diagnostic tests for detection of subclinical ketosis, defined as a serum beta-hydroxybutyrate (BHBA) concentration >or=1400 micromol/L. On 16 d over a 5-mo period, samples of serum, milk, and urine were collected on a large dairy facility from cows of all parities between 2 and 15 DIM. The sample proportion of subclinical ketosis was 7.6% (n = 859 samples from 545 cows). The KetoCheck powder (Great States Animal Health, St. Joseph, MO) detecting acetoacetate in milk samples was very specific (99%) but poorly sensitive (41%). Respective sensitivities and specificities of the Ketostix strip detecting acetoacetate in urine samples (Bayer Corporation, Elkhart, IN) were 78 and 96% with a cut-off point of "small", or 49 and 99% with a cut-off of "moderate." The KetoTest strip (Sanwa Kagaku Kenkyusho Co. Ltd., Nagoya, Japan) using milk samples had a sensitivity and specificity of 73 and 96% with a cut-off of 100 micromol of BHBA/L or 27 and 99% with a cut-off of 200 micromol of BHBA/L. On average, use of the Ketostix at the "small" cut-off point or the KetoTest at 100 micromol/L would result in no more than 3 or 4 false positives per 100 cows screened, with prevalence levels ranging from 5 to 30%, whereas the number of false negatives would range from one false negative at 5% prevalence to 7 or 8 false negatives at 30% prevalence. Either the Ketostix or KetoTest strips would provide acceptable results for screening individual cows on commercial dairies to detect subclinical ketosis. Over this prevalence range, the KetoCheck powder test would have limited application as a screening test. Despite only one false positive per 100 animals screened, false negatives resulting from screening with the KetoCheck test would be too frequent, ranging from 3 false negatives at 5% prevalence to 18 at 30% prevalence in a population of 100 tested cows. Finally, given their relative imprecision, use of any of these individual cowside tests to estimate herd prevalence must be done cautiously, especially when only a small number of animals are sampled.

Urinary acetoacetate or capillary beta-hydroxybutyrate for the diagnosis of ketoacidosis in the Emergency Department setting.

OBJECTIVES: We compared the semiquantitative measurement of acetoacetate using urinary dipsticks with the bedside quantitative fingerprick measurement of the principal ketone bodies 3-beta-hydroxybutyrate, for the diagnosis of ketoacidosis. METHODS: This is a one year retrospective study of patients who presented with hyperglycemia levels of 250 mg/l or greater in the Emergency Department setting. We compared the sensitivity, specificity, and predictive value of ketonuria and ketonemia for the diagnosis of ketoacidosis (urine or blood ketone bodies, blood bicarbonates <20 mmol/l, anion gap >16 meq/l) in a sample of patients for whom the levels of ketone bodies in the blood and urine as well as serum electrolytes were available. RESULTS: We studied 355 hyperglycemic patients. The median time between arrival and dipstick testing was 21 min, and was greater than 2 h in more than 10% of cases. Comparison between ketonuria and ketonemia was performed in 173 patients (6% with diabetic ketoacidosis). Ketonuria equal to or less than one cross or a 3-beta-hydroxybutyrate value lower than 3 mmol/l enabled ketoacidosis to be excluded (negative predictive value 100%). At two-cross cutoff points for ketonuria and at the 3 mmol/l cutoff point for ketonemia, the two tests had the same sensitivity (100%), but the specificity of 3-beta-hydroxybutyrate (94%) was significantly higher (P<0.0001) than that of ketonuria (77%). The best positive predictive value for ketonemia was obtained at the 5 mmol/l cutoff point (100%) and for ketonuria at the three-cross cutoff point (26%). At the three-cross cutoff point for ketonuria and at the 5 mmol/l cutoff point for ketonemia, the two tests had the same negative likelihood ratio (0.1), but the positive likelihood ratio of 3-beta-hydroxybutyrate (infinite) was higher than that of ketonuria. CONCLUSION: The measurement of 3-beta-hydroxybutyrate in capillary blood is faster and more effective than the use of dipsticks in the urine to detect ketoacidosis in the Emergency Department setting.

Glycerol kinase deficiency: follow-up during 20 years, genetics, biochemistry and prognosis.

AIM: To follow two children with isolated glycerol kinase deficiency (GKD) with severe symptoms into adulthood. METHODS: The patients were followed during approximately 20 y and interviewed about symptoms, diet and physical activity. Fasting provocations, bicycle ergometer tests, dietary registrations, enzyme and mutation analysis were performed by standard protocols. RESULTS: The activity of glycerol kinase (GK) in fibroblasts was <10% of reference. One case had a deletion of exon 17, the other a mutation in exon 7 of the GK gene (601 A-->G). Both mothers were heterozygotes. Two maternal male cousins in one of the families were hemizygotes without symptoms. Tests performed in childhood documented pronounced sensitivity to fasting and physical exercise, whereas such tests at 23 and 31 y of age were essentially normal but with pronounced ketonaemia. After puberty, the boys had no hypoglycaemic symptoms and now report no problems with their condition; thus, their phenotype has changed over time. CONCLUSION: The greater importance of glycerol as a gluconeogenetic substrate in children than in adults may explain the episodes in young patients with GKD, often elicited by catabolic stress. With meals at frequent intervals, access to glucose and avoidance of strenuous sports, the prognosis is good for a normal adult life of a young child with isolated GKD and symptoms of hypoglycaemia.

The mitochondrial acetoacetyl-CoA thiolase (T2) deficiency in Japanese patients: urinary organic acid and blood acylcarnitine profiles under stable conditions have subtle abnormalities in T2-deficient patients with some residual T2 activity.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism affecting isoleucine and ketone bodies in the catabolic process. Mutation analysis and expression analysis of mutant cDNAs have facilitated the division of T2-deficient patients into two groups: those with null mutations in either allele (group 1) and those with mutation(s) retaining some residual T2 activity in at least one of two mutant alleles (group II). Among 5 Japanese T2-deficient patients, GK01 belonged to group I and the other patients (GK19, GK19B, GK30 and GK31) to group II. As we have suggested previously, the severity of ketoacidotic episodes in the group II patients was similar to that in the group I patient. However, the urinary organic acid and blood spot acylcarnitine profiles under stable conditions differed between the two groups. The group I patient had typical profiles for the T2 deficiency. In contrast, in all four patients in group II, tiglylglycine was not or was only faintly detected and the 2-methyl-3-hydroxybutyrate levels were less than the cutoff value. Their tiglylcarnitine levels were within the normal range and 2-methyl-3-hydroxy-, butyrylcarnitine was detected just around the cutoff value in our newborn screening pilot test. Hence, these analyses under stable conditions are not reliable for diagnosing the T2 deficiency in the group II patients. The T2 deficiency (group II) can be misdiagnosed as normal if these analyses are performed under nonepisodic conditions and possibly during the newborn screening for inborn errors of metabolism.

A novel pyrroline-5-carboxylic acid and acetoacetic acid adduct in hyperprolinaemia type II.

BACKGROUND: From investigations of a child with hyperprolinaemia type II, we demonstrated in vitro that pyridoxal phosphate forms a novel adduct with a proline metabolite, pyrroline-5-carboxylic acid, through Claisen condensation. Studies indicated that this was a previously unsuspected generic reaction of aldehydes and some ketones. We have subsequently found the acetoacetic acid adduct in both plasma and urine from the affected child. METHODS: Mixtures of acetoacetic acid and pyrroline-5-carboxylic acid were co-incubated at pH 7.4 and 37 degrees C, dried, or extracted and dried, derivatised and analysed by gas chromatography/mass spectrometry (GC/MS). Urine and plasma from the child were analysed. RESULTS: Fourteen new peaks were found in derivatised pyrroline-5-carboxylic acid/acetoacetic acid co-incubates. From accurate molecular mass data, the four largest peaks were probably diastereoisomers of tri-trimethylsilyl (tri-TMS) derivatives of alcohol adducts formed by Claisen condensation. Eight other peaks were mono- and di-trimethylsilyl derivatives of the adduct and a decarboxylated product. The adduct was demonstrated unequivocally in the child's acute urine and traces in plasma. CONCLUSIONS: Pyrroline-5-carboxylic acid forms an adduct with acetoacetic acid, which was present in urine of a sick child with hyperprolinaemia type II. Evidence suggests it formed in vivo. The biological significance of this novel reaction of aldehydes and ketones merits investigation.

Accuracy of serum beta-hydroxybutyrate measurements for the diagnosis of diabetic ketoacidosis in 116 dogs.

The aim of this study was to evaluate the accuracy of serum beta-hydroxybutyrate (beta-OHB) measurements for the diagnosis of diabetic ketoacidosis (DKA) in dogs. One hundred sixteen diabetic dogs were prospectively enrolled in the study: 18 insulin-treated (IT) diabetic dogs that had a positive urine ketone test and 88 untreated, newly diagnosed diabetic dogs. Venous blood gas tensions and pH, serum glucose and urea nitrogen (SUN), and electrolyte (Na+, Cl-, and K+) and urine acetoacetate (AA) concentrations were measured concurrently with serum beta-OHB concentrations. On the basis of laboratory findings, the patients were assigned to I of 3 groups: diabetic ketoacidosis (n = 43); diabetic ketosis (DK, n = 41); and nonketotic diabetes (NDK, n = 31). Serum beta-OHB concentrations differed significantly (P < .001) among the study groups. Although marked differences in beta-OHB concentrations were found, a considerable overlap exists between the distributions of dogs with DK and those with DKA. The overall accuracy of beta-OHB determination as a diagnostic test for DKA, determined by the area under the receiver operating characteristic (ROC) curve, was 0.92. In the 1.9- to 4.8-mmol/L range, serum beta-OHB determination sensitivity varied from 100 to 35.7%, whereas specificity varied from 39 to 100%. The cutoff value of 3.8 mmol/L showed the best equilibrium between specificity (95%), sensitivity (72%), and likelihood ratio (14.8). We concluded that the quantitative measurement of serum beta-OHB may be a potential tool for diagnosing and monitoring ketosis and ketoacidosis in diabetic dogs.

Maleylacetoacetate isomerase (MAAI/GSTZ)-deficient mice reveal a glutathione-dependent nonenzymatic bypass in tyrosine catabolism.

In mammals, the catabolic pathway of phenylalanine and tyrosine is found in liver (hepatocytes) and kidney (proximal tubular cells). There are well-described human diseases associated with deficiencies of all enzymes in this pathway except for maleylacetoacetate isomerase (MAAI), which converts maleylacetoacetate (MAA) to fumarylacetoacetate (FAA). MAAI is also known as glutathione transferase zeta (GSTZ1). Here, we describe the phenotype of mice with a targeted deletion of the MAAI (GSTZ1) gene. MAAI-deficient mice accumulated FAA and succinylacetone in urine but appeared otherwise healthy. This observation suggested that either accumulating MAA is not toxic or an alternate pathway for MAA metabolism exists. A complete redundancy of MAAI could be ruled out because substrate overload of the tyrosine catabolic pathway (administration of homogentisic acid, phenylalanine, or tyrosine) resulted in renal and hepatic damage. However, evidence for a partial bypass of MAAI activity was also found. Mice doubly mutant for MAAI and fumarylacetoacetate hydrolase (FAH) died rapidly on a normal diet, indicating that MAA could be isomerized to FAA in the absence of MAAI. Double mutants showed predominant renal injury, indicating that this organ is the primary target for the accumulated compound(s) resulting from MAAI deficiency. A glutathione-mediated isomerization of MAA to FAA independent of MAAI enzyme was demonstrated in vitro. This nonenzymatic bypass is likely responsible for the lack of a phenotype in nonstressed MAAI mutant mice.