Effect of glucose, fructose, sorbitol and amino acid solutions employed in clinical medicine on the development of liver regeneration after partial hepatectomy

The authors studied the effect of glucose (G), fructose (F), sorbitol (So), 4% Nutramin S Spofa (NuS) and Nutramin C Spofa (NuC) on development of liver regeneration. NuS is used clinically to improve a negative nitrogen balance, while the amino acid composition of NuC has a beneficial effect on the ornithine cycle and thus helps to normalize the NH3 level in patients with liver failure. Glucose seems to be the best substance for an optimal onset of liver regeneration. Its onset in rats given NuC was slower than in animals given NuS, but 48 h after partial hepatectomy there was no difference between NuC and NuS as regards the total DNA content and 96 h after the operation there was no difference as regards either the number of mitoses or the number of liver cells. In addition to reducing the plasma ammonia level, therefore, NuC also has a favourable effect on the development on liver regeneration.     

人肝刺激因子对实验性急性肝功能衰竭小鼠的保护作用

取健康孕妇水囊引产的4～6个月龄的胎儿肝脏,按LaBreque法提取肝刺激因子(hHSS)。将hHSS注入肝部分切除的大鼠体内,用~3H-TdR掺入肝DNA测定其生物学活性。给昆明种小鼠腹腔注射D-半乳糖胺(D-Gal)以造成急性肝功能衰竭,观察hHSS对这种急性肝衰的保护效应,结果如下:①hHSS明显降低D-Gal所致的小鼠死亡率(P<0.01);②hHSS显著降低D-Gal所致sGPT和sGOT水平增高(sGPT 208.2±26.1比103.6±21.2 U/100ml;sGOT 502.6±65.4比287.8±39.4 U/100m1);③hHSS明显降低D-Gal所致的小鼠肝组织丙二醛水平增高(330.4±38.9比115.5±43.8nmol/100mg蛋白质);④光镜和电镜的组织学观察表明hHSS明显减轻D-Gal对肝组织的损害。上述实验结果表明,hHSS对D-Gal所致的急性肝衰具有明显的保护作用,其机制可能是保护肝细胞正常代谢和防止膜脂质过氧化。     

Collagen cross-linking. Effect of D-penicillamine on cross-linking in vitro.

D-Pencillamine is believed to inhibit collagen cross-link biosynthesis by forming thiazolidine rings with lysyl-derived aldehydes that are intermediates in bifunctional cross-link synthesis. Recently, we showed that aldehyde biosynthesis catalyzed by lysyl oxidase occurs after the onset of fibril formation and that nascent aldehydes form Schiff-base cross-links rapidly in fibrils. This suggested that the accessibility of D-penicillamine to most aldehydes formed during cross-link synthesis might be limited. To study this, reconstituted chick bone collagen fibrils were incubated in vitro with highly purified lysyl oxidase and D-penicillamine. As reported in previous studies in vivo, allysine content increased and polyfunctional cross-link synthesis decreased with D-penicillamine. However, the concentration of bifunctional cross-links increased rather than decreased due to a 2-fold increase in N6:6'-dehydro-5,5'-dihydroxylysinonorleucine. Hydroxyallysine, an intermediate in formation of this Schiff base, decreased. A time study indicated that allysine levels increased primarily after the bulk of Schiff base synthesis. These results indicate that D-penicillamine does not inhibit bifunctional cross-link synthesis as previously suggested. Its principal effect is to block synthesis of polyfunctional cross-link products from Schiff base cross-link precursors and to cause accumulation of these precursors. This effect may be due to interference with the close molecular packing required for polyfunctional cross-link synthesis. These results also suggest a mechanism for the relative insensitivity of tissues such as bone with high hydroxylysine content to D-penicillamine. In this study, D-penicillamine caused selective accumulation of allysyl and not hydroxyallysyl residues. In bone as opposed to soft tissues, hydroxyallysyl residues are intermediates in synthesis of almost all cross-links.     

Effect of realimentation after several days' pure carbohydrate intake on DNA synthesis in regenerating rat liver

The authors studied the effect of realimentation after several days' isolated glucose or fructose intake on DNA synthesis in liver regenerating after partial hepatectomy (PH) (65-70%) or after carbon tetrachloride (CCl4) poisoning 1.5 ml/kg. Two days before PH or the administration of CCl4 and two days after, the experimental rats were given glucose (50% solution) of fructose (50% solution) as the only source of energy. Rats with PH were then fed for one day on a standard laboratory diet (25 cal% protein) or a high protein diet (81 cal% protein). Rats with CCl4 liver damage were fed for one day on the standard laboratory diet only. In the rats given glucose, liver DNA synthesis and the total amount of these nucleic acids in the liver 48 hours after CCl4 administration was lower than in the controls or the rats given fructose. In all the experimental groups (PH and CCl4), stimulation of liver DNA synthesis was observed after one day's realimentation. The total DNA content of the liver of rats with PH rose markedly during realimentation. The experiments indicate that the regenerative activity of damaged liver can be influenced by the nutritional regimen.     

Influence of D-penicillamine on the formation of elastin and its cross-links.

The amount of insoluble elastin and its content of desmosine cross-links were investigated in aortas of chick embryos, to which D-penicillamine was administered on the 6th or 14th--16th day of incubation. D-Penicillamine was shown to alter the formation and maturation of elastin. Using lower doses (less than 50 mg) the weight of pooled aortic elastin is higher as compared with controls (related to 1 mg of elastin or to total weight of elastin). Increased isodesmosine:desmosine ratio in these samples indicates that this elastin is very young. On the other hand, a high dose of D-penicillamine (100 mg) decreased the content of elastin and also of its desmosine cross-links. The authors explain their findings by counteraction of two factors due to administration of penicillamine: the increased solubility of "insoluble elastin", and the decreased cross-link formation.     

Glycogen synthesizing function of hepatocytes in rats with liver cirrhosis after treatment with chorionic gonadotropin]

Using rat liver hepatocytes, methods of cytofluorimetry (Kudryavtseva et al., 1974) and biochemistry were applied to comparative studies of the total glycogen content, including its labile (LF) and stable (SF) fractions, and activities of glucose-6-phosphatase, glycogen phosphorylase and glycogensynthetase in these. The liver hepatocytes were examined in norm, and under conditions of CCl4 poisoning of rats, both 6 months after a chronic poisoning, and 1, 3 and 6 months following poisoning cessation. All the experimentally poisoned rats were divided into two conventional groups: rats of one group received, apart from poisoning, a complex treatment with chorionic gonadotropin (CG); the other group rats received, no treatment. The material used for examination was obtained from serial functional biopsies of each experimental animal. It has been shown that under cirrhosis the content of the total glycogen in hepatocytes increased by 3 times, and that of its SF even by 9.7 times. The treatment with CG for 1 month resulted in its reducing to the norm, and 3 to 6 months treatments normalized contents of both the glycogen fractions. In the group of non-treated rats no similar changes were registered. Besides, in the cirrotic rats the activity of glucose-6-phosphatase was shown to increase by 4 times. After CG treatment it was seen to decrease by 3 times. Thus, CG may be regarded as an optimum and more effective agent for restoring abnormalities in cirrotic liver, compared to some other stimulating factors, such as hepatectomy (Kudryavtseva et al., 1996) or rich-carbohydrate diet (Kudryavtseva et al., 1998).     

Effect of partial hepatectomy on the glycogen level in hepatocytes in the portal and central lobule zones in cirrhotically-changed rat liver

Using cytophotometric method, the content of glycogen was studied in hepatocytes of the portal and central zones of a liver lobule in norm, in cirrhosis, and 1, 3, and 6 months after a partial hepatectomy of the normal and cirrhotic rat liver. As we showed earlier, glycogen content in cirrhotic liver hepatocytes rose 2-3-fold, along with obvious impairment of glycogen metabolic heterogeneity in these. In cirrhotic liver glycogen dominates in the central zone, whereas in norm more glycogen is observed in the portal one. The objective of this study was to find out to what degree a partial hepatectomy of cirrhotic liver may promote recovery of the metabolic glycogen heterogeneity in hepatocytes. Glycogen was determined in hepatocytes, using a quantitative variant of PAS-reaction on sections of the material obtained from serial supravital punctate liver biopsies. Glycogen amount in hepatocytes of different liver lobule zones was determined by an image analyzer technique that allows to bring together the cytophotometric analysis of the substance with its localization in a particular liver lobule. Results of these studies have shown that a partial hepatectomy of cirrhotic liver promotes restoration of the hepatocyte metabolic heterogeneity in the liver lobule.     

Effects of dietary molybdenum, sulfur and zinc on the excretion and tissue accumulation of trace elements in sheep fed palm kernel cake-based diets

Twelve male 8-month-old lambs were used in a 6-month feeding experiment to determine the effects of dietary Mo, Mo + S and Zn supplements on the body retention and tissue accumulation of dietary Cu, Zn and Fe. The lambs were divided into four groups of three lambs each and each group was fed ad libitum one of four diets. A control diet was based on palm kernel cake (PKC) and grass hay. Three additional diets were the control supplemented with either Mo or Mo+S or Zn. At 3 months of the experiment, feces and urine were collected and sampled for 6 days. At the end of the experiment (6 months), blood was sampled and then the sheep were slaughtered. The liver and kidney were removed and sampled for chemical analysis. In comparison with the control, each dietary supplement decreased (P<0.05) the Cu concentration in the liver, but only the Mo+S supplement decreased it to a safe range of below 350 μg/g dry matter. This was accompanied by the body retention of dietary Cu of 24.6%, 6.7%, 2.5% and 6.5% for the control, Mo, Mo+S and Zn treatments, respectively. The blood plasma concentration of Cu was decreased (P<0.05) by the Zn supplement, but was not affected by other supplements (P>0.05). It was concluded that from the supplements tested, only Mo+S appeared to be effective in reducing the retention and liver accumulation of the dietary Cu to prevent chronic Cu toxicity in sheep fed PKC-based diets.     

Hydroxyurea Could Be a Good Clinically Relevant Iron Chelator

Our previous study showed a reduction in serum ferritin of β-thalassemia patients on hydroxyurea therapy. Here we aimed to evaluate the efficacy of hydroxyurea alone and in combination with most widely used iron chelators like deferiprone and deferasirox for reducing iron from experimentally iron overloaded mice. 70 BALB/c mice received intraperitonial injections of iron-sucrose. The mice were then divided into 8 groups and were orally given hydroxyurea, deferiprone or deferasirox alone and their combinations for 4 months. CBC, serum-ferritin, TBARS, sTfr and hepcidin were evaluated before and after iron overload and subsequently after 4 months of drug therapy. All animals were then killed. Iron staining of the heart and liver tissue was done using Perl’s Prussian Blue stain. Dry weight of iron in the heart and liver was determined by atomic absorption spectrometry. Increased serum-ferritin, TBARS, hepcidin and dry weight of iron in the liver and heart showed a significant reduction in groups treated with iron chelators with maximum reduction in the group treated with a combination of deferiprone, deferasirox and hydroxyurea. Thus hydroxyurea proves its role in reducing iron from iron overloaded mice. The iron chelating effect of these drugs can also be increased if given in combination.     

The effects of diabetes with hyperlipidemia on P450 expression in APA hamster livers

The effect of chronic hyperglycemia and hyperlipidemia induced by streptozotocin (SZ) on the expression of P450 in the liver of APA hamsters was studied in this experiment. No effect on the total activity of P450 was seen in SZ-induced diabetic hamsters throughout the experimental period. At 1 and 6 months after SZ-injection, the levels of CYP1A, 2C6, and 3A of SZ-injected hamsters were much lower than those of age-matched control hamsters. CYP2B expression tended to decrease and CYP2E1 and 4A expression tended to increase in SZ-injected hamsters, although the results were not significant. At 3 months after SZ-injection, however, no significant difference between SZ-injected and normal hamsters was seen in these P450 isozymes. On the other hand, CYP2C11 expression was slightly depressed in SZ1M and SZ6M, and almost equivalent to control hamsters in SZ3M. Immunohistochemistry by the use of each isozyme antibody revealed that SZ-induced diabetes affected the localization of CYP2C6, 3A, and 4A in the hepatic acinus. The expression of CYP2C6 and 3A was depressed mainly in the periportal region of the acinus, and CYP4A expression was induced mainly in the perivenous region by SZ-induced diabetes. On the other hand, the expression pattern of CYP1A, 2B, 2C11, and 2E1 were not affected. These results demonstrate that the effects of SZ-induced diabetes on hepatic P450 differ for each isozyme in APA hamsters and also differ from those of other experimental diabetic animals, including golden hamsters.     

Effect of long-term diet enrichment with selenium, vitamin E and vitamin B15 on the degree of fatty infiltration of the liver

In a two-year experiment on 190 Wistar rats the effects were studied of the aging process and diet enrichment with selenium, vitamin E and vitamin B15 (pangamic acid) on the degree of fatty infiltration of the liver determined histochemically with Oil Red O. The degree of fatty infiltration of the liver was assessed by the method of quantitative analysis using a computer image analyser Quantimet 720. System 30 (Cambridge Instruments). The process of aging of the animals was associated with increasing fatty infiltration of the liver. Selenium had a two-phase effect on the degree of fatty infiltration: in the first 12 months of selenium administration (0.1 ppm of sodium selenite per 100 g of the diet) fatty infiltration of the liver was decreasing, and after 18 months of the experiment this effect disappeared and the degree of fatty infiltration was not different from that in the control group. Contrary to this, vitamin E administration 6 mg/100 g of the diet increased the degree of fatty infiltration during the first 12 months. After 18 months a reverse effect appeared with inhibition of the progression of fatty infiltration. Thus the two-phase effect of vitamin E was a reverse of selenium effect. Addition of vitamin B15 to the diet (2.5 mg/100 g of diet) increased the degree of fatty infiltration of the liver which was maintained at a stable level throughout the whole experiment, i.e. 12-18 months.     

D-penicillamine-induced granulomatous hepatitis in brown Norway rats

The mechanism of idiosyncratic drug reactions (IDRs) remains poorly understood. D-penicillamine treatment is associated with a wide range of autoimmune reactions including liver injury. An animal model which utilizes brown Norway (BN) rats has been used to investigate the mechanism of D-penicillamine-induced IDRs because it mimics the autoimmune reactions that occur in humans. The purpose of this study was to investigate the type of liver injury that results from D-penicillamine treatment in BN rats. We had previously noted that D-penicillamine caused histological changes in the liver, but there was no increase in alanine transaminase (ALT), and we assumed that there was no significant injury. However, we subsequently discovered that D-penicillamine inhibits the ALT assay. In the present study, we found that treatment of BN rats with a low doses of D-penicillamine (10 or 15 mg/day) resulted in a mild increases in glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities; however, this was not associated with histological changes. A higher dose of D-penicillamine (20 mg/day) resulted in 63 % of the rats developing a skin rash, and these rats had elevated serum GLDH and SDH levels with histopathological changes characteristic of granulomatous hepatitis. This included large clusters of leukocytes in the form of granulomas that contained neutrophils, macrophages, and CD8 T cells. These changes did not occur in the rats that did not get sick. This model may be a good model to investigate the characteristics of drug-induced granulomatous hepatitis.     

Platelet aggregation and thromboxane A2 release in primary biliary cirrhosis and effect of D-penicillamine treatment

Platelet function was assessed in 28 patients with primary biliary cirrhosis (PBC), of whom 10 were receiving D-penicillamine. Patients not on D-penicillamine treatment had platelet aggregation similar to that in the healthy control group; the group treated with D-penicillamine showed significantly enhanced platelet aggregation in response to threshold doses of adrenaline and collagen but not ADP. Median thromboxane B2 production was also higher in D-penicillamine treated patients than in controls or untreated patients; this difference did not reach statistical significance. The addition of D-penicillamine in vitro to platelet rich plasma from normal subjects was shown to enhance adrenaline- and collagen-induced platelet aggregation. Abnormalities of platelet function in PBC patients did not correlate with serum cholesterol concentration or with liver function tests but were related to the stage of disease. The present study emphasises the need to consider the aetiology, disease stage and type of treatment when assessing platelet function and prostanoid release in liver disease.     

Radiation injury in rat lung. IV. Modification by D-penicillamine

To determine whether D-penicillamine, known to reduce fibrosis in irradiated rat lung (W. F. Ward, A. Shih - Hoellwarth , and R. D. Tuttle , Radiology 146, 533-537, 1983), also ameliorates radiation injury in the pulmonary endothelium, we measured angiotensin-converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) production in the lungs of penicillamine-treated (10 mg/day, po, continuous after irradiation) and untreated rats from 2 weeks to 6 months after a single dose of 25 Gy of 60Co gamma rays to the right hemithorax. Both ACE and PLA activity in the irradiated right lung of untreated rats decreased dramatically between the 1st and 2nd months after exposure, then reached a plateau through 6 months at approximately 25 and 50% of the normal level, respectively. For the first 2 months after irradiation, penicillamine-treated animals exhibited significantly (P less than 0.05) higher activities of both ACE and PLA than did untreated rats. From 3 to 6 months after irradiation, however, the only significant drug effect on these enzymes was a 25% increase in PLA activity at 6 months. PGI2 production by the irradiated lung of untreated rats increased continuously, and at 6 months was approximately 10 times higher than normal. Penicillamine significantly (P less than 0.05) reduced this hypersecretion, and at 6 months after irradiation, PGI2 production by the lungs of drug-treated rats was only half that of untreated animals. In contrast, the drug had no significant effect on enzyme activities in the lungs of sham-irradiated rats. Thus the antifibrotic agent D-penicillamine delays the onset of radiation-induced enzyme dysfunction in the pulmonary endothelium. In addition at 6 months after irradiation, the lungs of penicillamine-treated rats exhibit 25% more PLA activity and only half as severe a hypersecretion of PGI2 as do the lungs of untreated animals. The drug is most effective in ameliorating endothelial damage during the first 2 months after irradiation, preceding the development of interstitial fibrosis. However, the effect of this penicillamine regimen on pulmonary endothelial function is not as large as its effect on collagen accumulation in irradiated rat lung.     

An investigation into copper catalyzed D-penicillamine oxidation and subsequent hydrogen peroxide generation

D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.     

Molecular localization of copper and zinc in rat fetal liver in dietary and drug-induced copper deficiency

The teratogenicity of copper deficiency is well known, but underlying mechanisms have not been delineated. One method of studying the biochemical lesions of copper deficiency is the use of chelating drugs with different chemical characteristics. The teratogenicity of a copper deficient diet and of diets containing either D-penicillamine or triethylenetetramine is quite different, although all three diets result in decreased fetal liver copper levels. Feeding D-penicillamine can result in decreased fetal liver zinc, while feeding triethylenetetramine can result in increased fetal liver zinc. The effect of these three diets on fetal liver copper and zinc molecular localization was determined. Gel filtration showed that fetal liver copper and zinc in controls was localized in 3 fractions with MWs of > 50,000 (H), 30,000 (I) and 8–10,000 (L). Independent of dietary treatment, as liver copper diminished, copper was missing first from the L peak, then the I peak and with severe deficiency, from the H peak. Drug induced increases and decreases in fetal liver zinc were reflected in the L peak. These data suggest that the absolute levels of copper in the liver of the term fetus determines the distribution of the element among its binding ligands.     

Biochemical examination on the increased superoxide dismutase-like plasma substance observed in a state of acute gastric mucosal lesions

When experimental acute gastric mucosal lesions were produced in guinea pig by water-immersion and restraint stress, superoxide dismutase (SOD)-like substance in the plasma increased. On analysis by gel filtration, it was shown that the molecular weight of the increased SOD-like plasma substance was about 130,000, and even after treatment with trypsin, 84% of this substance remained. Since the molecular weight of intracellular SOD is about 40,000, it seems that this substance is similar to extracellular SOD, located on the endothelial cell-surface, as previously reported by Marklund et al. Our results suggest that in the presence of acute gastric mucosal lesions, SOD-like plasma substance is not identical to intracellular SOD, which derived from cell destruction by stress or free radical-induced microvascular damage or by hemolysis. Furthermore, this substance may itself work as a scavenger of free radicals generated under conditions, such as these described in the present experiment.     

D-penicillamine-induced cleft palate in mice

The teratogenic potential of the lathyrogen, D-penicillamine (DP), was assessed in pregnant mice, especially with respect to its ability to produce cleft palate. The dosage and the duration of treatment as they relate to the induction of cleft palate were also studied. Two different doses of DP were administered orally for either 5 or 4 consecutive days during the critical period of palatal closure. D-penicillamine (DP) at a dose level which does not have any apparent maternal toxic effects produced cleft palate in the offspring, and this teratogenic effect depended more upon the duration of treatment than the dosage administered. Inhibitory effects on the formation of bone matrix were observed at the base of the palatal shelf. It is suggested that DP is potentially an osteolathyrogenic agent. The mechanism of induction of cleft palate in DP-treated mice was explored by histological studies using light microscopy. Delayed elevation of the palatal shelves was observed and is considered to be the cause of the induction of cleft palate. No other external malformations could be detected in DP-treated fetuses.     

Role of the functional state of the liver RES in the pathogenesis of toxic liver injury

In rats to which E. coli endotoxin (250 micrograms/kg i.p.) was administered 24 h before they were given tetrachlormethane (CCl4) (1.5 ml/kg intragastrically), stimulation of liver DNA synthesis was observed during the first 48 h after administration of the hepatatoxin. In experimental rats to which prodigiosan (a Serratia marcescens polysaccharide, 250 micrograms/kg i.p.) was administered 24 h before CCl4 (1.5 ml/kg i.p.), liver damage 24 h after CCl4 poisoning was expressed less--judging from the size of liver necrosis and the size of glycogen-free zones in the liver lobules than in the controls. To elucidate the role of activated macrophages in the induction of liver resistance to CCl4, liver injury caused by this hepatotoxin was compared after the pre-administration of protein extract from the Kupffer cells or hepatocytes of prodigiosan-stimulated rats. In rats given the larger dose of Kupffer cell extract (6 mg/ml i.p.), the necrotic foci formed after the administration of CCl4 were significantly smaller. The results confirm the conception that liver macrophages participate in the development of resistance to CCl4.     

Capacitation of hamster spermatozoa with the divalent cation chelators D-penicillamine,L-histidine,and L-cysteine in a protein-free culture medium

By using a chemically defined (protein-free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D-penicilla-mine, L-histidine, and L-cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1–2 × 10⁶ sperm/ml) for 3, 4, or 6 hr at 37°C in 5% CO₂ in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein-free) containing 10 mM sodium lactate, 100 μM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 μM D-penicillamine, 100 μM hypotaurine, and 1.0 μM epinephrine). The low control sperm preincubation medium consisted of TLP-PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP-PVA). The experimental preincubation medium was TLP-PVA with additional D-penicillamine (125 or 500 μM), or L-histidine (10, 100, or 1,000 μM) or L-cysteine (25, 75, or 125 μM). After preincubation, sperm were coincubated (2 × 10⁴ sperm/ml) with cumulus-free hamster eggs in TALP-PVA ± additional D-penicillamine, L-histidine, or L-cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D-penicillamine (500 μM: range of mean penetration values, 53.6%–84.3%), L-histidine (100 μM: range of mean values, 24.8%–56.3%) or L-cysteine (75 μM: 51.3%) in the absence of exogenous protein.